AND-Gated Allosteric Protein-Based Switches

Abstract

The present invention relates to an improved protein-based biosensor suitable for the detection of one or more target molecules in a sample. The biosensor is fully reversible with a dynamic range suitable for analytical and diagnostic applications. The biosensor of the present invention can be used in synthetic biology, for example in the construction of artificial cellular or extracellular signaling networks.

Description

[Technical field]

[0001] The present invention relates to an improved biosensor. In particular, the present invention relates to an improved protein-based biosensor suitable for detecting one or more target molecules in a sample. The biosensor of the present invention may also be relevant in the field of synthetic biology, such as for constructing artificial cellular or extracellular signaling networks. The biosensor is fully reversible with a dynamic range suitable for analytical and diagnostic applications. [Background technology]

[0002] The construction of artificial protein switches with selectable input and output parameters is an important goal in synthetic biology and biotechnology. Protein biosensors have applications in clinical and industrial analysis, especially in diagnostics at the point of need. Direct conversion of analyte binding to a quantitative signal is an unmet need in this field for a simple, wash-free, homogeneous assay format. Furthermore, artificial protein-based switches that interconvert different biochemical signals also enable the construction of biochemical pathways with non-natural proteins for metabolic, cellular and biological engineering.

[0003] There are several competing approaches to constructing protein biosensors, all of which aim to create an off state of the desired reporter domain that can be reversed by the selected analyte. Previously described approaches to constructing protein-based biosensors include domain splitting, generating reversible autoinhibition, and inserting regulatory domains. Regulatory domain insertion provides direct, reversible coupling between ligand binding and reporter activity, but relies on a small ligand-binding domain with a global conformational change large enough to tune the activity of the reporter. Previously described biosensors have addressed this issue by using peptide receptor domains with large conformational changes and then applying protein engineering to couple the local concentration of the peptide ligand to the local concentration of the analyte. Such protein-based biosensors are described, for example, in WO 2016 / 191812, WO 2018 / 073588, and WO 2019 / 207356.

[0004] The key parameters that determine the usefulness of a biosensor are selectivity, sensitivity, dynamic range and response rate. Dynamic range is particularly important as it determines the signal-to-noise ratio and the precision with which measurements of target analytes can be made. The construction of protein switches with a practically relevant dynamic range is far from trivial and often requires extensive optimization of constructs generated by screening. Given the ensemble nature of allostery and the equilibrium nature of ligand binding, one would typically expect a trade-off between the dynamic range and reversibility of a system. Thus, systems that dislike one of the states (such as split proteins) would be expected to exhibit a wide dynamic range but poor / slow reversibility. For example, in one exemplary system, a cysteine ​​bond was introduced into the linker between the calmodulin regulatory domain insert and the glucose dehydrogenase reporter domain. Formation of a disulfide bond upon structural rearrangement of the receptor domain increased the dynamic range from 4-fold to almost 100-fold, but at the expense of reversibility.

[0005] This application addresses these problems. Described herein is an approach for the construction of fully reversible protein switches with very large dynamic range and fast activation times. This is achieved by constructing an integrated protein AND gate. Summary of the Invention

[0006] The present invention relates to improved reporter proteins, such as enzymes, that can be used in biosensors.In particular, the improved enzymes are particularly suitable for detecting target molecules in physiological conditions, such as biological samples.The improved enzymes contain multiple heterogeneous amino acid sensor domains, are fully reversible, and have improved sensitivity, enhanced dynamic range and fast response time, making them particularly suitable for detecting target molecules that are typically at low concentrations in physiological conditions, and allow simultaneous detection of multiple target molecules.

[0007] Thus, the present invention provides a reporter protein comprising a first heterologous amino acid sequence responsive to binding of a first regulatory factor portion and a second heterologous amino acid sequence responsive to binding of a second regulatory factor portion, wherein binding of the first regulatory factor portion to the first heterologous amino acid sequence and binding of the second regulatory factor portion to the second heterologous amino acid sequence reversibly modulates the activity of the reporter protein.

[0008] Also provided is a biosensor comprising a reporter protein as described herein and one or more (e.g., at least one) regulator moieties as described herein (e.g., a first regulator moiety as described herein and a second regulator moiety as described herein). Also provided as described herein. Also provided herein is a method of detecting one or more target molecules, comprising contacting a reporter protein, or a biosensor, or a composition, or a kit as described herein; with a sample under conditions suitable for detecting the presence or absence of one or more target molecules in the sample.

[0009] The invention further relates to (i) a method for diagnosing a disease or condition in an organism, comprising contacting a reporter protein, biosensor, composition, or kit described herein with a sample obtained from the organism under conditions suitable for detecting the presence or absence of one or more target molecules in the sample, where the presence or absence of the one or more target molecules in the sample indicates whether the organism has or is at risk of having the disease or condition; (ii) a method for monitoring one or more target molecules in an organism, comprising contacting a reporter protein, biosensor, composition, or kit described herein with a sample obtained from the organism under conditions suitable for detecting and / or quantifying the presence or absence of one or more target molecules in the sample; (iii) a method for monitoring a metabolite of interest in an organism, comprising contacting a reporter protein, biosensor, composition, or kit described herein with a sample obtained from the organism under conditions suitable for detecting and / or quantifying the presence or absence of one or more target molecules in the sample; (iv) a method of monitoring a metabolic product of interest comprising expressing a reporter protein or biosensor as described herein in an organism under conditions suitable for detecting and / or quantifying a metabolite of interest in the organism, optionally wherein the organism is a bacterial cell; or (iv) a method of assaying for a protein-protein or protein-small molecule interaction comprising contacting a reporter protein, biosensor, composition or kit as described herein with a sample under conditions suitable for detecting the presence or absence of an interaction between binding moieties B1' and B1" and / or an interaction between binding moieties B2' and B2"; or an interaction between binding moieties B1' and B1" and target molecule TM1 and / or an interaction between binding moieties B2' and B2" and target molecule TM2.

[0010] Also provided is a detection device comprising a cell or chamber containing a reporter protein or biosensor as described herein. Also provided is one or more nucleic acids encoding the reporter protein or biosensor as described herein. Also provided is one or more expression vectors comprising one or more nucleic acids operably linked to one or more promoters. Also provided is a host cell comprising the reporter protein, biosensor, or one or more nucleic acids or one or more expression vectors as described herein. Also provided is a method for expressing the reporter protein or biosensor as described herein in a host cell, comprising incubating the host cell as described herein under conditions suitable for expression of the reporter protein or biosensor.

[0011] Further provided is a method for converting a constitutively active enzyme into an enzyme whose catalytic activity is reversibly regulated depending on the presence of one or more target molecules, comprising: (a) generating a library of single insertion enzyme mutants by inserting a heterologous amino acid sequence responsive to binding of a regulator moiety at several different positions within the enzyme sequence, and selecting the single insertion enzyme mutants that exhibit a change in catalytic activity upon binding of the regulator moiety; and (b) generating a library of double insertion enzyme mutants by inserting an additional heterologous amino acid sequence responsive to binding of the regulator moiety at a second site within the enzyme sequence, and selecting the double insertion enzyme mutants that exhibit a change in catalytic activity in the presence of the regulator moiety and have an increased dynamic range compared to the corresponding single insertion enzyme mutant; optionally wherein the change in catalytic activity occurs within less than 10 minutes, optionally within less than 5 minutes. [Brief description of the drawings]

[0012] [Figure 1]Design of artificial allosteric enzymes by domain insertion. (A) Schematic of a constitutively active reporter protein domain. (B) Construction of a chimeric protein by insertion of a receptor domain into a reporter protein (exemplified by calmodulin). (C) Activation of reporter protein activity by interaction of the chimera with a ligand (exemplified by a calmodulin-binding peptide) that induces a conformational change in the receptor domain. (D) Schematic of the enzyme cascade for the conversion of trehalose to gluconolactone by trehalase (Tre) and PQQ-glucose dehydrogenase (PQQ-GDH). [Figure 2A] Construction and activity analysis of trehalase-CaM chimeras. (A) Schematic diagram of chimeric Tre constructs. [Figure 2B] Construction and activity analysis of trehalase-CaM chimeras. (B) Schematic diagram of Tre-CaM activation by calmodulin-binding peptide (CaM-BP). [Figure 2C] Construction and activity analysis of trehalase-CaM chimeras. (C) Structure of trehalase (PDB: 2jg0) shown in ribbon representation with insertion positions shown as balls. [Figure 2D] Construction and activity analysis of trehalase-CaM chimeras. (D) Activity analysis of a solution of 50 nM Tre-CaM mutants mixed with 50 nM GDH in the presence of 50 mM trehalose, 0.6 mM PMS as electron mediator and 60 μM DCPIP reporter dye in the presence (broken trace) or absence (solid trace) of 0.5 μM M13 CaM-BP. The identities of the mutants are indicated by their insertion positions. [Diagram 3]Construction of Tre chimeras with two CaM regulatory domains. (A) Schematic of the design process based on the combination of single insertion variants identified using the screening approach outlined in Figure 2D. (B) Activity analysis of a 50 nM solution of Tre G104_S440 CaM chimera in the presence or absence of 0.5 μM M13 CaM-BP. Assays were performed as in Figure 2D. (C) As in (B) but using TreG104_P321 CaM chimera. (D) Activity of a 50 nM solution of Tre G104_P321 chimera in the presence of different concentrations of M13 CaM-BP peptide. (E) Fitting of data from the titration shown in (D) to an apparent Kd value of 65 nM. (F) Ribbon representation model of Tre G104_P321 CaM chimera. Calcium ions are displayed as balls and M13 peptide is displayed as ribbon. [Figure 4]Using the 2CaM-Tre switch module to construct a small molecule AND-gate biosensor with a wide dynamic range. (A) Schematic of a rapamycin biosensor based on an AND-gate switch, where the 2CaM-Tre switch module is tagged with FKBP domains at the C-terminus and N-terminus. The activation domain consists of an FRB fusion with the low affinity CaM-BP peptide. Addition of rapamycin drives the association of the components and activation of trehalase activity. (B) Titration of 100 nM 2FKBP-Tre-2CaM and 200 nM FRB-CaM-BP with increasing concentrations of rapamycin. (C) Fitting of the titration data shown in (B) to an apparent Kd of 89 nM. (D) Construction of a biosensor based on an AND-gate Tre switch tuned by two inputs. The Tre-2CaM switch module is tagged with FKBP on the N-terminus and a cyclophilin domain on the C-terminus. As activators, FRB-CaM-BP and fusions of calcineurin A and B (CalA / B) with CaM-BP are used. (E) Activity of the two input system shown in (D) in the presence of rapamycin, cyclosporin A or their mixture. In the experiment, a 100 nM solution of FKBP-Tre-2CaM-cyclophilin was mixed with 100 nM FRB-CaM-BP and 100 nM CalA / B-CaM-BP and 50 nM wt GDH in the presence or absence of 350 nM rapamycin, cyclosporin or their mixture, and the change in absorbance of the DCIP dye was recorded over time. (F) To confer an additional level of regulation to the system shown in D, the AND-gate biosensor shown in (A) was combined with a binary GDH biosensor of cyclosporin A. (G) The cyclophilin (CyP) domain-fused CaM-GDH chimera is activated by CalA / B fused to CaM-BP in the presence of cyclosporin A.(H) Time trace of GDH activity of the system shown in (F) and (G) in which 100 nM FKBP-Tre-2CaM-FKBP was mixed with 200 nM FRB-CaM-BP, 5 nM CaM-GDH-cyclophilin domain, and 30 nM CalA / B-CaM-BP in the presence or absence of 250 nM rapamycin, cyclosporin A, or a mixture thereof. [Figure 5A] Construction of β-lactamase single insert CaM chimera (BLA-CaM). (A) Schematic diagram showing the β-lactamase (BLA) assay. BLA cleaves the β-lactam ring nitrocefin, which can be detected by a color change from yellow to red using, for example, a wavelength of λ=482 nm (wavelengths of λ=486 nm and λ=480 nm can also be used). [Figure 5B] Construction of the β-lactamase single-insert CaM chimera (BLA-CaM). (B) Structure of β-lactamase in ribbon representation (PDB: 3gmw) with the calmodulin insertion site displayed as a ball. [Figure 5C] Construction of β-lactamase single insert CaM chimeras (BLA-CaM). (C) Activity of purified BLA-CaM chimeras in the presence (broken line) or absence (solid line) of saturating concentrations of M13 peptide. The insertion position is indicated by the number and letter indicating the deleted amino acid. [Figure 6]Construction of a β-lactamase double-inserted CaM chimera (2CaM-BLA). (A) Activity analysis of a solution of 250 nM BLA-CaM 41G in the presence of 50 μM nitrocefin and in the presence (broken line) or absence (solid line) of 1 μM M13 CaM-BP. (B) Activity of the reaction mixture from (A) with 25 nM BLA-CaM 41G preincubated for the indicated period before adding nitrocefin. (C) Activity analysis of a solution of 250 nM BLA-CaM 197E in the presence of 50 μM nitrocefin and in the presence (broken line) or absence (solid line) of 1 μM M13 CaM-BP. (D) Activity of the reaction mixture from (C) with 25 nM BLA-CaM 197E preincubated for the indicated period before adding nitrocefin. (E) Schematic of a chimeric BLA biosensor containing two CaM modules. (F) Activity of 250 nM BLA-CaM 41G_197E in the presence of 50 μM nitrocefin in the presence (broken line) or absence (solid line) of 1 μM M13 CaM-BP. (G) Activity of reaction mixture from (F) with 10 nM BLA-CaM 41G_197E preincubated for the indicated period before adding nitrocefin. (H) Plot of activity of 10 nM wild-type β-lactamase, single CaM inserted β-lactamase and double CaM CaM41G and CaM197E inserted β-lactamase in the presence of saturating concentrations of M13 CaM-BP. (I) Plot of maximum activity at 10 nM of wild-type β-lactamase, double CaM CaM41G and CaM197E inserted β-lactamase and single inserted CaM-BLA 253G chimera with a large dynamic range of 207-fold. [Figure 7]Rapamycin biosensor based on 2CaM-BLA switch. (A) Schematic of the two-component biosensor for rapamycin based on the BLA-CaM 41G_197E switch unit. (B) Activity analysis of solutions of 25 nM BLA-2CaM-2FKBP fusion protein and 100 nM FRB-CaM-BP (low affinity calmodulin binding peptide) in the presence of 50 μM nitrocefin and increasing concentrations of rapamycin. (C) Fitting of the data shown in (B) to a quadratic equation results in an apparent Kd value of 11 nM. (D) Activity analysis of solutions of 25 nM BLA-2CaM-2FKBP fusion protein, 100 nM FRB-CaM-BP and 250 nM rapamycin incubated for the indicated periods before addition of 50 μM nitrocefin. [Figure 8] Tacrolimus biosensor based on 2CaM-BLA switch. (A) Schematic of the two-component biosensor for tacrolimus based on the BLA-CaM 41G_197E switch unit. (B) Activity analysis of solutions of 25 nM BLA-2CaM-2FKBP fusion protein and 100 nM calcineurin A / B-CaM-BP (a low affinity calmodulin binding peptide) in the presence of 50 μM nitrocefin and increasing concentrations of tacrolimus. (C) Fitting of the data shown in (B) to a quadratic equation results in an apparent Kd value of 14 nM. (D) Activity analysis of solutions of 25 nM BLA-2CaM-2FKBP fusion protein, 100 nM FRB-CaM-BP and 250 nM tacrolimus incubated for the indicated periods before addition of 50 μM nitrocefin. [Figure 9]Methotrexate biosensor based on 2CaM-BLA switch. (A) Schematic of a two-component biosensor for methotrexate (MTX) based on the BLA-CaM 41G_197E switch unit. (B) Arrows indicate time traces of absorbance change for the following solutions: 10 nM BLA-2CaM 41G197, 10 nM BLA-2CaM 41G197 and 1 μM M13 CaM-BP (added at the 10 min mark), 10 nM VHH-BLA-2CaM 41G197-VHH mixed with 100 nM nanoCLAMP-BP, 10 nM VHH-BLA-2CaM 41G197-VHH mixed with 100 nM nanoCLAMP-BP, and 250 nM methotrexate (added at the 10 min mark). (C) Activity of 10 nM VHH-BLA-2CaM 41G197-VHH and 100 nM nanoCLAMP-BP solutions supplemented with the indicated concentrations of methotrexate. (D) Fitting of the data shown in (C) to a quadratic equation gives an apparent Kd value of 7.3 nM. (E) Activity analysis of solutions of 10 nM VHH-BLA-2CaM 41G197-VHH and 100 nM nanoCLAMP-BP, 250 nM MTX incubated with the indicated organs prior to addition of 50 μM nitrocefin. (F) Plot of the data shown in (E) fitted to a single exponential function. [Figure 10]Use of the developed 2CaM-BLA biosensor for the construction of a clinically useful MTX assay. (A) Photograph of the Beckman AU-480 clinical chemistry analyzer used to test the binary MTX biosensor based on the β-lactamase dual CaM CaM41G and CaM197E switch module. (B) Time trace of 50 μM nitrocefin supplemented with 20 mM Tris pH 7.4, 10 nM VHH-BLA-2CaM 41G197-VHH and 100 nM nanoCLAMP-BP containing 100 mM NaCl buffer, and the indicated concentrations of methotrexate standards in human serum (Abbot Architect MTX calibration kit). (C) Plot of the data shown in (B) fitted to a linear function. (D) Analysis of serum samples of 21 patients undergoing MTX therapy using the 2CaM-BLA MTX biosensor (X-axis) or the Abbot Architect immunochemistry station (Y-axis). [Figure 11] Construction of a glucose dehydrogenase (GDH) AND gate biosensor. (A) Activation of GDH-CaM 48N by CaM-BP, activity analysis of a solution of 10 nM GDH-CaM 48N in the presence of 0.6 mM PMS as an electron mediator and 60 μM DCPIP reporter dye in the presence or absence of 1 μM M13 CaM-BP. (B) Activation of GDH-CaM 212N by CaM-BP, activity analysis of a solution of 10 nM GDH-CaM 212N in the presence of 0.6 mM PMS as an electron mediator and 60 μM DCPIP reporter dye in the presence or absence of 1 μM M13 CaM-BP. (C) Activation of GDH-CaM 48N_212N by CaM-BP. Activity assay of a solution of 10 nM GDH-CaM 48N_212N in the presence of 0.6 mM PMS as electron mediator and 60 μM DCPIP reporter dye in the presence or absence of 1 μM M13 CaM-BP. [Figure 12]Schematic of biosensor design requiring only one ligand binding domain. (A) Biosensor in which binder 2 is operably linked to two CaM-BPs, each with low affinity for calmodulin. (B) Biosensor design in which ligand binder 2 is operably linked between two fusion proteins, each containing wild-type CaM-BP and a calmodulin domain with low affinity for CaM-BP. Addition of ligand (star) results in interaction between ligand binder 1 and ligand binder 2 with the ligand. Colocalization of ligand binder 2 fusion protein with reporter domain (black box) results in relocation of CaM-BP to the high affinity calmodulin module on the reporter domain, thereby resulting in activation of the biosensor. [Figure 13] Use of two CaM-reporter units to monitor the activity of a protease. (A) Schematic showing a fusion protein in which CaM-BP is caged to a calmodulin mutant with low affinity for CaM-BP. The dashed line represents the protease cleavage site. (B) As in (A) but with unit duplication resulting in higher binding activity of the component and reducing background activity. (C) Caged unit with duplicate cages designed to reduce background activity of the caged peptide. (D) As in (C) but with a protease binding domain designed to increase the local concentration of the protease, thereby increasing the specificity of the system for a particular protease and increasing the rate of protein degradation. [Figure 14] Electrochemical detection of β-lactamase activity. Cyclic voltammograms of 0.5 mM nitrocefin (dashed line) and the product of its enzymatic hydrolysis (solid line). Conditions: carbon paper-based electrode (immersion 0.5 × 0.5 cm); scan rate 50 mV / s vs. Ag / AgCl (3 M KCl); 100 mM PBS, pH 7.2; β-lactamase (150–300 U / mL); enzyme reaction time 10 min; all experiments were performed in the dark at room temperature. [Figure 15]Analysis of expression of BLA-CaM fusion proteins in E. coli and ability of transgenic bacteria to survive ampicillin. (A) Plasmid map of modified pACYDuet vector designed to constitutively express a protein of interest in E. coli cells. (B) Optical density of DH5-α E. coli strain transformed with pACYDuet-BLA-CaM253G grown overnight in LB medium containing chloramphenicol at a concentration of 34 μg / ml and ampicillin at a concentration of 100 μg / ml in the presence of CaM-BP at the indicated concentrations. (C) Agar plates containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol inoculated with cells transformed with pACYDuet-BLA-CaM253G vector. After plating, CaM-BP at the indicated concentrations was spotted on the plate at the indicated positions and the plate was incubated at 37° C. overnight. Bacterial growth appears as dark spots. [Figure 16] Use of CaM-BP to control ampicillin resistance in bacteria transformed with BLA-CaM fusions. Agar plates containing 34 μg / ml chloramphenicol (left) or 100 μg / ml ampicillin and 34 μg / ml chloramphenicol (right) were plated with DH5-α cells transformed with the indicated constructs and grown overnight at 37° C. [Figure 17A] Constitutive expression of BLA-2CaM fusion proteins in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (A) Plasmid map of the modified pACYDuet modified BLA-2CaM 41G 197E vector designed to express a protein of interest in DH5-α E. coli strain. [Figure 17B] Constitutive expression of BLA-2CaM fusion protein in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin (B) Agar plate containing 34 μg / ml chloramphenicol and seeded with cells transformed with the pACYDuet-modified-BLA-2CaM 41G 197E vector. [Figure 17C]Constitutive expression of BLA-2CaM fusion protein in E. coli and analysis of the ability of transgenic bacteria to survive on ampicillin. (C) Agar plate containing 34 μg / ml chloramphenicol and 100 μg / ml ampicillin and seeded with cells transformed with the pACYDuet-modified-BLA-2CaM 41G 197E vector. [Figure 17D] Constitutive expression of BLA-2CaM fusion protein in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (D) Optical density of DH5α cells transformed with pACYDuet-modified-BLA-2CaM 41G 197E vector grown overnight in LB medium containing chloramphenicol at a concentration of 34 μg / ml and ampicillin at a concentration of 100 μg / ml in the presence of the indicated concentrations of CaM-BP. [Figure 17E] Constitutive expression of BLA-2CaM fusion proteins in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (E) Agar plates containing 100 μg / ml ampicillin and 34 μg / ml chloramphenicol seeded with cells transformed with pACYDuet-modified-BLA-2CaM 41G 197E vector. After plating, the indicated concentrations of CaM-BP were spotted onto the plates at the indicated locations and the plates were incubated overnight at 37° C. Bacterial growth appears as dark spots. [Figure 18A] Inducible expression of BLA-2CaM fusion proteins in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (A) Plasmid map of the modified PET28a-BLA-2CaM 41G 197E vector designed for inducible expression of a protein of interest in BL21(DE3) E. coli cells. [Figure 18B]Inducible expression of BLA-2CaM fusion proteins in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (B) Optical density of BL21(DE3) E. coli cells transformed with PET28a-BLA-2CaM 41G 197E grown overnight in LB medium containing kanamycin at a concentration of 50 μg / ml and ampicillin at a concentration of 100 μg / ml with or without 1 mM IPTG in the presence of the indicated concentrations of CaM-BP. [Figure 18C] Inducible expression of BLA-2CaM fusion proteins in E. coli and analysis of the ability of transgenic bacteria to survive ampicillin. (C) SDS-PAGE gel loaded with cells from the experiment shown in (B). Lane 1 is loaded with purified BLA-2CaM 41G 197E protein. Agar plates containing 100 μg / ml ampicillin and 50 μg / ml kanamycin seeded with cells transformed with the PET28a-BLA-2CaM 41G 197E vector. After plating, the indicated concentrations of CaM-BP and 1 mM IPTG were spotted onto the plates at the indicated locations and the plates were incubated overnight at 37° C. Bacterial growth appears as dark spots. [Figure 19] Emission scans of mNeon chimeric fluorescent proteins with one or two calmodulin domains. 1 μM solutions of mNeon-CaM chimera (small grey diamonds) and mNeon-2CaM (black balls) were scanned for emission while keeping the excitation wavelength constant at 500 nm ± 5 nm. The scans were repeated with the addition of 5 μM of the M13 calmodulin binding peptides mNeon-CaM chimera (grey triangles) and mNeon-2CaM (black diamonds). [Figure 20]Activity analysis of GDH affinity clamp single and double insertion chimeras. (A) Activity analysis of a 10 nM solution of a GDH-affinity clamp chimera with an affinity clamp inserted at position 46. Assays were performed in the absence (solid line) and presence (dashed line) of 1 μM RGS ligand peptide. Assays were performed as described in the legend to FIG. 11. (B) As in (A) but using a solution of a GDH-affinity clamp chimera with an affinity clamp inserted at position 54. (C) As in (A) and (B) but using a solution of a GDH-affinity clamp chimera with two affinity clamps inserted at positions 46 and 54. [Figure 21] Biosensors based on 2CaM-Tre and 2CaM-GDH switch modules are used to construct a biosensor system with a wide dynamic range regulated by two different inputs. (A) Schematic of an allosteric enzyme cascade regulated at two different nodes. Here, an AND-gated Tre A-based biosensor controlled by rapamycin is combined with an AND-gated GDH biosensor for methotrexate. (B) Activity plot of the enzyme cascade shown in A, where 100 nM FKBP-2CaM-Tre-FKBP was mixed with 200 nM FRB-CaM-BP, 20 nM 2CaM-GDH-2VHH domains and 100 nM nanoCLAMP-CaM-BP in the presence or absence of 250 nM rapamycin, methotrexate or their mixture. [Figure 22]Thermostabilized methotrexate biosensor based on 2CaM-BLA switch. (A) Activity of a 10 nM solution of 2CaM-BLA-41G 197E in the presence of a saturating concentration of M13 CaM-BP after incubation at the indicated temperatures for the following periods: 4° C.-3 h, 25° C.-3 h, 37° C.-1 h, 50° C.-30 min, 60° C.-10 min, and 80° C.-10 min. (B) As in A, but using the K55Q, S82A, G92D, T140K, H153R, V184A mutants of 2CaM-BLA-41G 197E. (C) Change in absorbance of 10 nM 2CaM-BLA-41G / 197E and its K55Q, S82A, G92D, T140K, H153R, and V184A mutants in the absence and presence of 1 μM CaM-BP. (D) Relative activity of 10 nM 2CaM-BLA-41G 197E and its K55Q, S82A, G92D, T140K, H153R, and V184A mutants in the presence of saturating concentrations of M13 peptide. (E) Activity of a 10 nM solution of a methotrexate biosensor based on 2VHH 2CaM-BLA-41G 197E K55Q, S82A, G92D, T140K, H153R, V184A mutant mixed with 100 nM nanoCLAMP-CaM-BP titrated with increasing concentrations of methotrexate. (F) Fitting of the plot of Kobs ratio from C to a quadratic equation gave a Kd value of 8.6 ± 1.8 nM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present invention relates to improved reporter proteins that can be preferably used in biosensors capable of detecting target molecules. Typically, reporter proteins comprise two or more (i.e., more than one, a plurality) heterologous amino acid sequences that act as sensor domains and undergo a conformational change upon binding of a regulator moiety. The two or more heterologous amino acid sequences can be the same or different sequences. Binding of the regulator moiety to the two or more heterologous amino acid sequences induces a conformational change in the two or more heterologous amino acid sequences that epistatically interact (i.e., have an additive effect) and impart a large conformational change to the reporter protein, switching it from an "OFF" state to an "ON" state or from an "ON" state to an "OFF" state. The reporter protein then produces a detectable signal that can be detected and / or quantified. By coupling the association of each of the heterologous amino acid sequences with its respective regulator moiety in a manner that is dependent on the presence of one or more target molecules, for example by linking the heterologous amino acid sequence and the regulator moiety to a binding moiety pair configured to bind to the target molecule, activation of the reporter protein can be dependent on the presence of one or more target molecules. The reporter proteins and biosensors described herein have the advantage of a wide dynamic range within clinically useful response times, and also allow the input of multiple different target molecules.

[0014] Thus, the present invention provides a reporter protein (e.g., an enzyme, fluorescent protein or binding domain, preferably an enzyme or fluorescent protein, most preferably an enzyme) comprising a first heterologous amino acid sequence responsive to binding of a first regulator moiety and a second heterologous amino acid sequence responsive to binding of a second regulator moiety, where binding of the first regulator moiety to the first heterologous amino acid sequence and binding of the second regulator moiety to the second heterologous amino acid sequence reversibly modulates (preferably reversibly activates) an activity of the reporter protein (e.g., catalytic activity of an enzyme, or fluorescence of a fluorescent protein, etc.). Typically, each heterologous amino acid sequence (i.e., the first and second heterologous amino acid sequences) is provided as an insertion within the amino acid sequence of the reporter protein (i.e., enzyme or fluorescent protein).

[0015] One advantage of the claimed reporter proteins and biosensors comprising them is the significantly improved dynamic range, while still maintaining the reversible regulation of the reporter protein's activity. The dynamic range of the reporter protein (preferably an enzyme) can be at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold or 150-fold. The dynamic range of the reporter protein is optionally at least 10-fold, preferably at least 20-fold. The dynamic range of the biosensor comprising the reporter protein described herein can be at least 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold or 150-fold. The dynamic range of the biosensor comprising the reporter protein described herein is optionally at least 10-fold, preferably at least 20-fold. The dynamic range provides a measure of the ratio between the maximum achievable signal and background activity of the biosensor / reporter protein under optimal conditions for detection. The larger the dynamic range, the better the signal-to-noise ratio of the biosensor / reporter protein, i.e., the more sensitive the biosensor / reporter protein, and the smaller the change in concentration of the target molecule that can be detected by the biosensor / reporter protein. The dynamic range of the biosensor / reporter protein of the present invention can be calculated by comparing the activity of the biosensor / reporter protein in the absence of the target molecule with the activity of the biosensor / reporter protein when the target molecule is present at a saturating concentration, i.e., a concentration of the target molecule that does not further increase the activity of the biosensor / reporter protein with further increase in concentration. Thus, the dynamic range (X) can be defined as the maximum possible signal level of the biosensor / reporter protein (i.e., in the presence of a saturating concentration of the target molecule, under optimal reaction and detection conditions) (B) divided by the background activity level (A), which is the signal level of the biosensor / reporter protein in the absence of the target molecule in the assay. Thus, the dynamic range X=B / A.For example, if the maximum possible signal level B is 60 and the background activity level A is 30, then the dynamic range X=B / A=60 / 30=2, i.e., a 2-fold dynamic range. For example, a 10-fold dynamic range may be provided by a biosensor with a background activity level of 1 and a maximum signal level of 10 (X=B / A=10 / 1=10-fold). The signal level may be defined as the observed reaction rate (i.e., the initial reaction rate) obtained by fitting the linear phase of the reaction curve.

[0016] definition The term "variant" as used herein (e.g., in connection with a reporter protein, heterologous amino acid sequence, or regulator moiety described herein) may refer to a functional fragment of an amino acid sequence, protein, or peptide of the invention, suitably retaining their relevant activity or function, catalytic activity, or binding activity, as appropriate. Variants may include amino acid sequences that include deletions or insertions compared to any of the amino acid sequences disclosed herein. Such deletions or insertions may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, or 1-5, 1-10, 1-20, 1-30, 1-40, or 1-50 amino acids in length, preferably 1-10 amino acids in length. Optionally, such deletions or insertions occur within a loop, or at the N-terminus or C-terminus. Variants may preferably include amino acid sequences that include mutations (i.e., substitutions, point mutations) relative to the corresponding wild-type amino acid sequence or the corresponding amino acid sequence disclosed herein. A variant may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mutations, or 1-5, 1-10, 1-15, 1-20, 1-30, 1-40 or 1-50 mutations, optionally 1-20, preferably 1-10, most preferably 1-5 mutations, relative to the corresponding wild-type amino acid sequence or to the corresponding reference amino acid sequence disclosed herein. For example, typically conservative amino acid variations may be made without an obvious or substantial change in function. For example, conservative amino acid substitutions may be tolerated if the charge, hydrophilicity, hydrophobicity, side chain "bulk", secondary and / or tertiary structure (e.g., helicity), binding of the modulator moiety, binding of the target molecule, fluorescence, enzymatic activity and / or inhibitory activity is not substantially altered or is altered to an extent that does not appreciably or substantially impair the function of the biosensor. Variants may include amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to any of the amino acid sequences disclosed herein.

[0017] The term "fragment" as used herein (e.g., in connection with a reporter protein, heterologous amino acid sequence, or regulator portion described herein) typically refers to a functional fragment of an amino acid sequence, protein, or peptide of the invention, suitably retaining its relevant activity or function, catalytic activity, or binding activity, as appropriate. Fragments are typically N-terminal and / or C-terminal truncations. Protein fragments may comprise up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, preferably up to 80%, 85%, more preferably up to 90%, or most preferably 95%-99% of the amino acid sequence disclosed herein. In some embodiments, a protein fragment may comprise up to 5, 10, 20, 40, 50, 70, 80, 90, 100, 120, 150, 180 200, 220, 230. 250, 280, 300, 330, 350, 400 or 450 amino acids of the amino acid sequence disclosed herein. In some embodiments, a protein fragment may comprise a deletion of 1-10, 1-20, 1-30, 1-40, 1-50, 1-75, 1-100, preferably 1-50, most preferably 1-100 amino acids relative to the wild-type sequence of the protein, typically the deletions occur at the N-terminus and / or C-terminus. The fragment is typically a functional fragment in that it retains an activity of interest (i.e., an activity relevant to the biosensors described herein) of the corresponding wild-type protein. The fragment may comprise a domain contained within the full-length protein, typically the protein domain contained within the fragment is the protein domain responsible for the activity of interest of the wild-type protein. Fragments may contain up to 5, 10, 20, 40, 50, 70, 80, 90, 100, 120, 150, 180 200, 220, 230. 250, 280, 300, 330, 350, 400 or 450 amino acids of an amino acid sequence disclosed herein.

[0018] "Sequence identity" as used herein can be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two protein / polypeptide / peptide or two nucleic acid / polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first or second sequence for optimal alignment). The amino acid or nucleotide residues at corresponding positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., homology %=number of identical positions / total number of positions*100), optionally with a penalty in the score for the number of gaps introduced and / or the length of the gaps introduced. Sequence identity is typically measured over the length of the shorter sequence.

[0019] Comparison of sequences and determination of percent identity between two sequences can be achieved using algorithms. Alignment can be generated over a certain portion of the aligned sequences that have sufficient identity but not over a portion that has a low degree of identity (i.e., local alignment). A non-limiting example of a local alignment algorithm utilized for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68 (modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77). Such an algorithm is incorporated into the BLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

[0020] Alignment can be optimized by introducing appropriate gaps and percent identity can be determined over the length of the aligned sequences (i.e., gapped alignment). To obtain gapped alignment for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. Alignment can be optimized by introducing appropriate gaps and percent identity can be determined over the entire length of the aligned sequences (i.e., full-range alignment). A non-limiting example of an algorithm utilized for full-range comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated in the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.

[0021] The sequence identity between two sequences is preferably determined using pairwise global sequence alignment, and alignment is calculated over the length of the sequence described herein (preferably over the length of the shorter sequence).Sequence identity can preferably be calculated using the Needleman-Wunsch alignment algorithm (e.g., implemented through online server EMBOSS Needle (EMBOSS: the European Molecular Biology Open Software Suite. (2000) Trends in genetics. 16 (6): 276-7), applying the following parameters: Matrix: DNAfull; gap open penalty: 10.00; gap extension penalty: 0.5; end gap penalty: false; end gap open penalty: 10.00; end gap extension penalty: 0.5.

[0022] The term "linker" as used herein (e.g., in connection with a reporter protein, a heterologous amino acid sequence, a binding moiety or a modulator moiety described herein) may be understood to mean any means of linking (i.e., joining or conjugating) two proteins, peptides or amino acid sequences together. A linker preferably comprises or consists of an amino acid sequence and may typically be 1-20, 1-15, 1-10 or 1-5 amino acids long, preferably 1-10 amino acids long. Typically, such linkers comprise glycine and serine residues, preferably at least 50%, 60%, 70%, 80%, 90% glycine and serine.

[0023] The term "species of", e.g., species of heterologous amino acid sequences, proteins, modulator moieties, binding moieties or target molecules, may be understood to refer to a type or variety of proteins / moieties. For example, the same species of proteins typically have the same or closely related amino acid sequences or contain the same protein domains. Thus, the same species of proteins may have at least 95% sequence identity, preferably at least 98% sequence identity, with each other when measured over the length of the shorter sequence. The same species of small molecules or ions may have the same chemical identity or the same or closely related structures, and are thus recognized as variants of the same molecular structure. For example, the same species of molecules may share a common molecular structure, such as at least 90%, preferably 95%, and most preferably at least 98% of the same molecular structure. A different species is any two proteins / molecules that are not recognized as the same species according to these definitions. A species may include multiple instances of a particular protein / molecule.

[0024] Reporter proteins The reporter protein may be any protein capable of producing or generating a detectable and / or measurable signal. The reporter protein may be any protein capable of producing or generating a detectable and / or measurable signal, which may be reversibly inactivated so that a conformational change in the structure of the reporter protein reduces or eliminates the production or generation of the detectable and / or measurable signal. The reporter protein may be an enzyme, a fluorescent protein or a binding domain. The reporter protein is preferably an enzyme or a fluorescent protein, most preferably an enzyme. The detectable and / or measurable signal may be, for example, an electron, a change in redox state, a proton (e.g., a change in pH), a color change, antibiotic resistance, luminescence, fluorescence and / or radioactivity. The detectable signal may be measured using electrochemical methods (e.g., FIG. 14), thus electrochemically measuring the hydrolysis of the enzyme substrate to form a product. A suitable electrochemical detection method may be measuring a cyclic voltammogram, for example under the conditions described in the description of FIG. 14, as will be well understood by those skilled in the art. Electrochemical methods can distinguish between substrates and products, and can optionally detect electrons generated during such reactions. Electrochemical methods can be capable of detecting electrons generated during the conversion of a substrate to a product. Electrochemical methods can be capable of being performed in biological fluids, such as serum. For example, β-lactamase activity can be detected using electrochemical methods to monitor the hydrolysis of the substrate nitrocefin.

[0025] Typically, an enzyme, when catalytically active, can react with or act on an enzyme substrate, thereby inducing a detectable signal. Non-limiting examples of suitable enzymes include trehalase, β-lactamase (ampicillin resistance protein), glucose dehydrogenase, oxidoreductase or carbonic anhydrase (e.g., flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH), NAD-dependent glucose dehydrogenase (NAD-GDH) or pyranose dehydrogenase (PDH)), aminoglycoside phosphotransferase (kanamycin resistance protein), α-amylase, carbonic anhydrase, β-galactosidase, glucose oxidase, lysozyme, malate dehydrogenase, peroxidase (e.g., horseradish peroxidase (HRP)), phosphatase (e.g., alkaline phosphatase), luciferase, transferase, ATPase, nuclease (e.g., ribonuclease), kinase, synthase. Preferably, the enzyme is trehalase, β-lactamase, glucose dehydrogenase, oxidoreductase or dehydrogenase (such as FADGDH or PDH), aminoglycoside phosphotransferase, α-amylase or carbonic anhydrase. Most preferably, the enzyme is trehalase, β-lactamase or glucose dehydrogenase. Non-limiting examples of suitable enzyme substrates include those that allow for the production of chromogenic, fluorescent, light (e.g. bioluminescent), electrical, pH, radioactive and other detectable signals, such as antibiotic resistance, when the enzyme is expressed in a suitable host cell, such as a microbial cell, a yeast cell, or preferably a bacterial cell.

[0026] Typically, a fluorescent protein can emit light at a specific detectable wavelength when active, usually upon excitation at a specific wavelength. Non-limiting examples of suitable fluorescent proteins include mNeon, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), Cherry, mCherry, mCFP, mTurquoise2, mOrange, mKate, dsRed, eqFP611, Dronpa, TagRFP, KFP, EosFP / IrisFP, Dendra, bacterial phytochrome (BphP)-based fluorescent proteins (see, e.g., Matlashov, ME, et al., Nat Commun 2020; 11, 239), or cyanobacteriochrome (CBCR)-derived fluorescent proteins (such as miRFP670nano, see, e.g., Oliinyk OS, et al., Nat Commun. 2019; 10(1): 279). The BphP and CBCR-based fluorescent proteins may include a cofactor for fluorescence (e.g., biliverdin). The fluorescent protein may be GFP or Cherry. The fluorescent protein may be mNeon, a BphP-based fluorescent protein, or a CBCR-based fluorescent protein. The fluorescent protein may be mNeon. The reporter protein may be the fluorescent protein mNeon, in which case the detectable signal is the fluorescence emission of mNeon. The mNeon protein preferably comprises a sequence having at least 90%, and optionally 100%, identity to the sequence of SEQ ID NO: 73, or a variant or functional fragment thereof. The variants and functional fragments typically retain fluorescent activity.

[0027] An enzyme is typically a protein that exhibits catalytic activity on a substrate molecule, thereby capable of producing a detectable signal. The reporter protein may be the enzyme trehalase, in which case the substrate may be trehalose and the catalytic activity may be the conversion of trehalose to glucose. In the presence of an oxidoreductase or dehydrogenase enzyme, such as GDH, preferably pyrroloquinoline quinone-GDH (PQQ-GDH), glucose is converted to gluconolactone, thereby producing electrons, in which case the detectable signal is the production of electrons. The trehalase preferably comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, and in some cases 100% identity to the sequence of SEQ ID NO: 1, or a variant or functional fragment thereof. The reporter protein may be the enzyme β-lactamase, where the substrate may be nitrocefin and the catalytic activity may be the cleavage of the β-lactam ring of nitrocefin, thereby resulting in a color change from yellow to red, and thus the detectable signal is a color change from yellow to red. The β-lactamase preferably comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, and optionally 100% identity to the sequence of SEQ ID NO: 3, or a variant or functional fragment thereof (e.g. SEQ ID NO: 4 or SEQ ID NO: 85). In some embodiments, the β-lactamase is a thermostable β-lactamase comprising a sequence having at least at least 98% or at least 99%, and optionally 100% identity to the sequence of, for example, SEQ ID NO: 85, and the reporter protein can be the enzyme GDH, preferably PQQ-GDH, in which case the substrate can be glucose and the catalytic activity can be the conversion of glucose to gluconolactone, thereby producing electrons, and thus the detectable signal is the production of electrons.The PQQ-GDH preferably comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, and optionally 100% identity to the sequence of SEQ ID NO: 5, or a variant or functional fragment thereof. The eGFP-GDH fusion protein may comprise a sequence having at least 90%, and optionally 100% identity to the sequence of SEQ ID NO: 78, or a variant or functional fragment thereof. The reporter protein may be the enzyme β-lactamase or the enzyme aminoglycoside phosphotransferase, in which case the substrate may be an antibiotic, such as ampicillin or kanamycin, respectively. The catalytic activity may be the cleavage of the β-lactam ring of an antibiotic, and when the β-lactamase or aminoglycoside phosphotransferase is expressed in a host cell, such as a bacterial cell, the detectable signal may be to confer antibiotic resistance to the host cell (β-lactamase confers ampicillin resistance, and aminoglycoside phosphotransferase confers kanamycin resistance). The aminoglycoside phosphotransferase preferably comprises a sequence having at least 90%, and optionally 100%, identity to the sequence of SEQ ID NO: 8, or a variant or functional fragment thereof. The reporter protein may be the enzyme carbonic anhydrase, in which case the catalytic activity is typically the interconversion of carbon dioxide to water, and bicarbonate to hydrogen ions, and thus the detectable signal is a change in pH. The carbonic anhydrase may comprise a sequence having at least 90%, and optionally 100%, identity to the sequence of SEQ ID NO: 10, or a variant or functional fragment thereof. The reporter protein may be the enzyme α-amylase, where the catalytic activity is typically the hydrolysis of polysaccharides (e.g., amylose, starch, amylopectin, glycogen), most usually the hydrolysis of α-1,4 glycosidic bonds to form intermediate oligosaccharides (e.g., dextrins), maltose and glucose. The α-amylase may comprise a sequence having at least 90%, and optionally 100% identity to the sequence of SEQ ID NO: 83, or a variant or functional fragment thereof.

[0028] The reporter proteins (preferably enzymes) of the invention comprise a first heterologous amino acid sequence responsive to binding of a first regulator moiety and a second heterologous amino acid sequence responsive to binding of a second regulator moiety. The reporter proteins of the invention may comprise more than one, optionally two or more, optionally a plurality of, heterologous amino acid sequences, each responsive to binding of a regulator moiety. The reporter proteins of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 heterologous amino acid sequences, optionally 1-5, preferably 1-3, most preferably 2 heterologous amino acid sequences, each responsive to binding of a regulator moiety.

[0029] The first heterologous amino acid sequence may be provided as an insertion in the amino acid sequence of the reporter protein, and the second heterologous amino acid sequence may be provided as an insertion in the amino acid sequence of the reporter protein. Preferably, the heterologous amino acid sequences are inserted at different positions in the amino acid sequence of the reporter protein. Each, preferably all, of the heterologous amino acid sequences may be provided (i.e., inserted) as an insertion in the amino acid sequence of the reporter protein. The insertion of two or more heterologous amino acid sequences into the amino acid sequence of the reporter protein typically reversibly inactivates the reporter protein, such that at least partial inactivation may be observed upon insertion of one heterologous amino acid sequence. Thus, the insertion of two or more heterologous amino acid sequences into the amino acid sequence of the reporter protein may have an additive or epistatic effect.

[0030] Binding of the regulator moiety to each (i.e., all) of the heterologous amino acid sequences reversibly regulates (preferably activates) the activity of the reporter protein (preferably the catalytic activity of the enzyme). Typically, binding of the first regulator moiety to the first heterologous amino acid sequence and the second regulator moiety to the second heterologous amino acid sequence reversibly activates the activity of the reporter protein. Typically, simultaneous binding of the regulator moiety at all of the heterologous amino acid sequences inserted into the reporter protein is necessary for full activation of the reporter protein. Thus, the reporter protein / enzyme is preferably engineered to be switchable from a state of reduced activity to a more active state depending on the binding of the regulator moiety. Typically, binding of the regulator moiety to each and every heterologous amino acid sequence is necessary to fully activate the reporter protein, and in the absence of the regulator moiety, the reporter protein typically does not exhibit any detectable activity.

[0031] It should be understood that wild-type activity cannot be conferred to the reporter protein by the attachment of a regulator moiety. Typically, a reporter protein is active if a detectable signal, such as electrons, protons, light, fluorescence, antibiotic resistance, or colored product, is produced and can be detected and / or measured under appropriate reaction conditions. If the reporter protein is an enzyme, the enzyme can be catalytically active if it can exhibit specific enzymatic activity on a substrate molecule to produce a detectable signal (e.g., electrons, protons, light, color change, colored product, antibiotic resistance, or fluorescence) under appropriate reaction conditions. If the reporter protein is a fluorescent protein, it can be active if it exhibits detectable fluorescence upon excitation at an appropriate wavelength or emission. As generally used herein, the term "inactive" can refer to a reporter protein that is substantially incapable of exhibiting a detectable and / or measurable signal under appropriate reaction conditions. As generally used herein, the term "catalytically inactive" can refer to an enzyme that is substantially incapable of exhibiting specific enzymatic activity on a substrate molecule under appropriate reaction conditions. Typically, the detectable signal produced (e.g., electrons, protons, light, color change, colored product, antibiotic resistance, or fluorescence) will be substantially less than that produced by a corresponding active reporter protein, or, if the reporter protein is an enzyme, compared to that produced by a corresponding catalytically active enzyme, e.g., a corresponding wild-type enzyme. Production of a detectable signal (e.g., electrons, protons, light, color change, colored product, antibiotic resistance, or fluorescence) may be completely absent for an inactive reporter protein, such as a reporter protein that includes two or more heterologous amino acid sequences.

[0032] The reporter proteins, preferably enzymes, described herein, and biosensors comprising them, produce a detectable signal (e.g., electrons, protons, light, color change, colored product, antibiotic resistance, or fluorescence) by reacting with a substrate molecule or excitation wavelength in response to binding, interaction, or other detection of one or more target molecules. In this context, react, reaction, or reacting with a substrate molecule means enzymatically converting a substrate molecule into one or more product molecules, the reaction producing a detectable signal, or the product molecules can be detected directly or indirectly (e.g., a glucose product produced by trehalase can be indirectly detected, for example, by using a PQQ-GDH enzyme to convert glucose to gluconolactone, thereby producing electrons, which can be detected). In this context, react, reaction, or reacting with an excitation wavelength means absorbing light of a particular excitation wavelength and emitting light of a different wavelength to produce a detectable fluorescent signal.

[0033] If the detectable signal is electrons, the biosensor can act as an electron donor, whereby electrons produced by the reaction can flow directly or through an electron shuttle (i.e., an electron mediator), such as, but not limited to, phenazine methosulfate or potassium ferrocyanide, thereby acting as an anode. The resulting change in potential between the anode and the cathode can be detected by an electron detector. If the reporter protein is an enzyme and the detectable signal is the production of electrons, the reporter protein or biosensor can be attached to an electrode. The mode of attachment can allow for direct electron transfer from the oxidoreductase or biosensor to the electrode. Typically, the biosensor or enzyme acts as an electron donor, whereby electrons produced by the reaction can flow directly to the electrode to form the anode. The electrode can be composed of gold, platinum, carbon nanotubes, or graphene. The enzyme or biosensor can be attached to the electrode surface using 1-pyrenebutanoic acid succinimidyl ester (PBSE) as a heterobifunctional linker, where the active ester group of the PBSE linker can react with the amino group of a lysine residue in the oxidoreductase or biosensor. The electrode can be a screen-printed electrode laminated with a dry mixture comprising the oxidoreductase and / or biosensor of the invention, preferably further comprising an electron mediator. The enzyme can be immobilized on the electrode via a modified cofactor (such as, for example, PQQ). The modified cofactor can be functionalized with a linker attached to the surface of the electrode, either covalently or non-covalently, for example, via an attachment group (e.g., via a pyrene-carbon nanotube interaction).

[0034] The reporter proteins, enzymes, fluorescent proteins, or biosensors described herein can be lyophilized (i.e., freeze-dried, cryodessicated), for example, by using typical low-temperature dehydration processes well known in the art. The reporter proteins, enzymes, fluorescent proteins, or biosensors described herein can be lyophilized and rehydrated (i.e., reconstituted) and retain their activity, i.e., do not exhibit significantly reduced activity after lyophilization and rehydration, compared to their activity before lyophilization. The reporter proteins, enzymes, fluorescent proteins, or biosensors described herein can be lyophilized, rehydrated, and stored at room temperature for at least 7 days and retain their activity.

[0035] Heterologous amino acid sequence Described herein is a reporter protein (preferably an enzyme) that comprises two or more heterologous amino acid sequences. Described herein is a reporter protein (preferably an enzyme) that comprises a first heterologous amino acid sequence that is responsive to the binding of a first regulator portion and a second heterologous amino acid sequence that is responsive to the binding of a second regulator portion, where the binding of the first regulator portion to the first heterologous amino acid sequence and the binding of the second regulator portion to the second heterologous amino acid sequence reversibly regulate the activity of the reporter protein. The reporter protein may further comprise one or more additional heterologous amino acid sequences.

[0036] The heterologous amino acid sequence may be provided as an insertion in the amino acid sequence of the reporter protein. Preferably, all of the heterologous amino acid sequences contained in the reporter protein are provided as an insertion in the amino acid sequence of the reporter protein. In some examples, the first heterologous amino acid sequence is provided as an insertion in the amino acid sequence of the enzyme, and the second heterologous amino acid sequence is provided as an insertion in the amino acid sequence of the reporter protein. However, fusion of the heterologous amino acid sequence at the N-terminus and / or C-terminus of the amino acid sequence of the reporter protein is also possible. Preferably, the reporter protein amino acid sequence and the two or more heterologous amino acid sequences (e.g., the first and second heterologous amino acid sequences) are present in or form at least part of a single contiguous amino acid sequence.

[0037] When provided as an insertion, the two or more (i.e., the first and second and any further) heterologous amino acid sequences are contiguous with the respective portions, subsequences or fragments of the reporter protein. The two or more heterologous amino acid sequences may be inserted at the same position within the amino acid sequence of the reporter protein, for example consecutively (i.e., one after the other, as a continuous sequence), with the C-terminus of one heterologous amino acid sequence linked consecutively to the N-terminus of the next heterologous amino acid sequence. However, preferably, each heterologous amino acid sequence is inserted into the amino acid sequence of the reporter protein at a different insertion position. The insertion is typically made at a position within the amino acid sequence of the reporter protein that allows the insertion without steric clashes that would prevent stable folding of the reporter protein. Preferably, the heterologous amino acid sequence is inserted into the amino acid sequence of the reporter protein in a solvent-exposed portion of the reporter protein. The heterologous amino acid sequence may be inserted into either an alpha helix, a beta sheet or a loop in the amino acid sequence of the reporter protein, preferably near the active site; typically in the order of insertion site location preference, with loops being most preferred, followed by beta sheets, followed by alpha helices being least preferred. Near the active site may be understood to mean any site where structural perturbation results in a conformational change in the conformation of the amino acid residues that make up the active site of the reporter protein (i.e., a site involved in activity that produces a detectable or measurable signal). Thus, the insertion may be made within 5 Å, 10 Å, 15 Å, 20 Å, 25 Å or within 50 Å, preferably within 20 Å, most preferably within 10 Å, of the nearest active site residue. Each heterologous amino acid sequence may be provided as an insertion in the amino acid sequence of the reporter protein flanked on both sides by linkers. Linkers may be added between each heterologous amino acid insertion and the sequence of the reporter protein to aid in tolerating the insertion. Similarly, when two or more heterologous amino acid sequences are linked in series, they may be connected via a linker. The linker may be as described herein.

[0038] The reporter protein may contain each heterologous amino acid sequence in a loop or turn region in the reporter protein structure that functionally allows the insertion of the heterologous amino acid sequence. The reporter protein may contain two or more heterologous amino acid sequences in one or more positions (such as loops or turns) in the reporter protein that contain one or more amino acid residues that affect the activity of the reporter protein. Thus, when the reporter protein is an enzyme, the heterologous amino acid sequence insertion may replace one or more residues that affect the substrate binding and / or catalytic activity of the enzyme, such that the catalytic activity of the enzyme is regulated by two or more heterologous amino acid sequences (e.g., a first heterologous amino acid sequence and a second heterologous amino acid sequence, and optionally further heterologous amino acid sequences). When the reporter protein is an enzyme, each heterologous amino acid sequence insertion may replace another residue that affects substrate binding and / or one or more residues that affect catalytic activity. When the reporter protein is an enzyme, each heterologous amino acid sequence insertion may prevent or reduce substrate binding to the enzyme and / or switch the enzyme to a state of reduced catalytic activity or catalytically inactive. Thus, when the reporter protein is a fluorescent protein, the heterologous amino acid sequence insertions may replace one or more residues that affect the structure of the fluorophore of the fluorescent protein, such that the fluorescence of the fluorescent protein is modulated by two or more heterologous amino acid sequences. When the reporter protein is a fluorescent protein, each heterologous amino acid sequence insertion may prevent or reduce the level of fluorescence that may be emitted by the fluorescent protein.

[0039] The heterologous amino acid sequences may be proteins. Preferably, all of the heterologous amino acid sequences are proteins. Typically, each of the heterologous amino acid sequences is a protein capable of undergoing a conformational change in response to binding of a regulator moiety. Each heterologous amino acid sequence may comprise one more domain (e.g., one or two domains) that undergoes a structural rearrangement (i.e., a conformational change) upon binding of the regulator moiety. Each heterologous amino acid sequence may comprise an unstructured or unfolded amino acid sequence that undergoes a structural rearrangement or conformational change upon binding of the regulator moiety, possibly generating one or more folded protein domains. Each heterologous amino acid sequence may be a protein that undergoes a structural rearrangement or conformational change upon binding of the regulator moiety that alters its interaction with the reporter protein. For example, binding of the regulator moiety may induce a structural rearrangement of the heterologous amino acid sequence to form a binding site for the reporter protein, such that the reporter protein and the heterologous amino acid sequence associate, thereby modulating the activity of the reporter protein. Preferably, each heterologous amino acid sequence is a protein that undergoes a structural rearrangement or conformational change upon binding of a regulator moiety that increases or decreases the distance in space between the N-terminus and C-terminus of the heterologous amino acid sequence. Thus, binding of a regulator moiety to a heterologous amino acid sequence may cause a conformational change in the structure of the heterologous amino acid sequence. Each heterologous amino acid sequence may be a different protein, but preferably each of the heterologous amino acid sequences is the same protein, or a functional variant or fragment thereof. For example, the first and second heterologous amino acid sequences may be different proteins, or preferably the same protein, or a functional variant or fragment thereof. The first heterologous amino acid sequence may be a first species of protein or a functional fragment thereof, and the second heterologous amino acid sequence may be a second species of protein or a functional fragment thereof. In some examples, at least one (preferably all) of the heterologous amino acid sequences is a calmodulin protein, or a variant or functional fragment thereof. In some examples, at least one (preferably all) of the heterologous amino acid sequences is an affinity clamp as further described herein.In some examples, at least one (preferably all) of the heterologous amino acid sequences is a solute-binding protein as further described herein. The first heterologous amino acid sequence can be a calmodulin protein, and the second heterologous amino acid sequence can be an affinity clamp. Preferably, all of the heterologous amino acid sequences (e.g., the first heterologous amino acid sequence and the second heterologous amino acid sequence, and optionally any further heterologous amino acid sequences) are each a calmodulin protein or a functional fragment thereof. In a preferred example, at least one, preferably each, of the heterologous amino acid sequences is a calmodulin protein, an engineered variant thereof, or a functional fragment thereof.

[0040] At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be a calcium-binding protein or a functional fragment thereof. At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be a calmodulin protein or a functional fragment thereof. At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be an affinity clamp (e.g., an ePDZ domain). At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be a solute-binding protein (SBP) (also known as a periplasmic binding protein (PBP) or Venus-fly trap protein, see, e.g., Edwards, KA, Talanta Open, 2021, 3:100038). At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be an SH3 domain. At least one, preferably all (e.g., the first and second) heterologous amino acid sequences may be an antibody. At least one, and preferably all (e.g., the first and second) heterologous amino acid sequences can be leucine zipper peptides. The reporter protein includes two or more heterologous amino acid sequences (e.g., the first and second heterologous amino acid sequences), and thus there can be a single species of heterologous amino acid sequence, each heterologous amino acid sequence being the same. Or, there can be multiple different species of heterologous amino acid sequences.

[0041] The reporter protein may comprise 2, 3, 4, 5, 6, 7, 8.9, 10 or more heterologous amino acid sequences. The reporter protein may comprise 2 to 10 heterologous amino acid sequences. The reporter protein may comprise 2 to 5 heterologous amino acid sequences. Preferably, the reporter protein may comprise 2 or 3 heterologous amino acid sequences, each of which may be the same or different. Most preferably, the reporter protein comprises two heterologous amino acid sequences, each of which may be the same or different. Thus, the reporter protein may comprise multiple different species of heterologous amino acid sequences, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more species of heterologous amino acid sequences. Preferably, the reporter protein comprises one or two different species of heterologous amino acid sequences. Most preferably, the reporter protein comprises a single species of heterologous amino acid sequence. Thus, in some preferred examples, the reporter protein comprises a first and a second heterologous amino acid sequence that are the same species of heterologous amino acid sequence, i.e. they are the same. The heterologous amino acid sequence may be selected from a calmodulin protein or a functional fragment thereof; an affinity clamp (eg, an ePDZ domain); an SBP; an SH3 domain; an antibody; a leucine zipper peptide; or a combination thereof.

[0042] Typically, at least one, and preferably all (i.e., the first and second, and optionally any further) heterologous amino acid sequences are amino acid sequences of calcium-binding proteins or functional fragments thereof. When at least one heterologous amino acid sequence is a calcium-binding protein or functional fragment thereof, the modulation of the activity of the reporter protein typically requires the presence of calcium ions. In a preferred example, at least one, and preferably all (i.e., the first and second, and optionally any further) heterologous amino acid sequences comprise or are preferably calmodulin proteins or functional fragments thereof. The calmodulin proteins or functional fragments thereof may be calcium-insensitive. The "calmodulin proteins" described herein may be any calmodulin proteins or domains and variants thereof previously described, including derivatives and variants of the specific calmodulin proteins or domains described herein. At least one, or preferably all, of the heterologous amino acid sequences may comprise or consist of a calmodulin protein comprising a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to any one of SEQ ID NO: 11, 12, 15 or 16, preferably SEQ ID NO: 11, or a variant or functional fragment thereof. At least one, or preferably each, of the heterologous amino acid sequences may comprise or consist of any one of SEQ ID NO: 11, 12, 15 or 16, preferably SEQ ID NO: 11, or a variant or functional fragment thereof. An exemplary wild-type calmodulin protein is shown in SEQ ID NO: 15. Variants and functional fragments are described herein and in this context typically retain calmodulin activity. For example, variants and functional fragments typically retain calcium binding activity and / or calmodulin-binding peptide (CaM-BP) binding activity, preferably both activities.The variants and functional fragments may not have calcium binding activity and / or may be calcium insensitive. Such variants and functional fragments typically retain "calmodulin binding peptide" binding activity. If a calmodulin protein or functional fragment thereof is calcium insensitive, it may bind calcium, but such calcium binding may not significantly affect the conformation of the calmodulin protein or functional fragment thereof. In some examples, at least one, and preferably each, of the heterologous amino acid sequences may comprise or consist of a calmodulin protein or functional fragment thereof that includes one or more modifications that result in a reduced binding affinity for calmodulin binding peptide (CaM-BP). Such calmodulin proteins with reduced binding affinity to CaM-BP may comprise or consist of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to SEQ ID NO: 16, or a variant or functional fragment thereof. Such calmodulin proteins with reduced binding affinity to CaM-BP may comprise or consist of SEQ ID NO: 16, or a variant or functional fragment thereof.

[0043] When at least one, or preferably all, of the heterologous amino acid sequences is a calmodulin protein or a functional fragment thereof, the activity of the reporter protein may be modulated in the presence of physiological calcium (i.e., by binding of a regulator moiety to each of the heterologous amino acid sequences). Thus, typically, it is the binding of the regulator moiety to the calmodulin protein that induces a conformational change in the calmodulin protein, rather than the binding of calcium. Thus, typically, the regulator moiety is not calcium. Physiological calcium concentrations are typically between 500 μM and 5 mM calcium (i.e., Ca 2+), preferably 1 mM to 2 mM calcium. In preferred examples, binding of the regulator moiety to each heterologous amino acid sequence reversibly regulates the activity of the reporter protein in the presence of at least 1 mM calcium, preferably 1 mM to 2 mM calcium. In some examples, changes in calcium concentration of 0.5 to 5 mM, preferably 1 mM to 2 mM, do not appreciably affect the activity of the reporter protein. Thus, the activity of the reporter enzyme is typically independent of calcium concentration when the calcium concentration is within physiological levels.

[0044] Typically, at least one, and preferably all (i.e., the first and second, and optionally any further) heterologous amino acid sequences are an affinity clamp amino acid sequence or a functional fragment thereof. In a preferred embodiment, at least one, and preferably all (i.e., the first and second, and optionally any further) heterologous amino acid sequences comprise or are preferably affinity clamps. The "affinity clamp" described herein may be any of the affinity clamps and variants thereof previously described. At least one, or preferably all, of the heterologous amino acid sequences may comprise or consist of an affinity clamp comprising a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to one of SEQ ID NO: 48 or 76, preferably SEQ ID NO: 76, or a variant or functional fragment thereof. At least one, or preferably each, of the heterologous amino acid sequences may comprise or consist of the sequence of SEQ ID NO: 76, or a variant or functional fragment thereof. The variants and functional fragments typically retain the ability to bind to an affinity clamp ligand, which may preferably be an RGS peptide ligand. An exemplary RGS peptide ligand may comprise or consist of the sequence of SEQ ID NO: 82. Thus, at least one, or preferably all (i.e., the first and second, and optionally any further) heterologous amino acid sequences may optionally comprise or consist of an affinity clamp comprising the sequence of SEQ ID NO: 76, which is capable of binding to a peptide ligand, which optionally comprises the sequence of SEQ ID NO: 82. Typically, binding of the peptide ligand to the affinity clamp induces a conformational change in the affinity clamp, an activity which is typically retained by variants and functional fragments.

[0045] regulator part A regulator moiety is any moiety that can be bound to a heterologous amino acid sequence as defined herein. Typically, binding of the regulator moiety to a heterologous amino acid sequence causes or induces a conformational change or structural rearrangement of the heterologous amino acid sequence. Thus, the nature or identity of the regulator moiety typically depends on the nature or identity of the heterologous amino acid sequence. Thus, when there are two different species of heterologous amino acid sequence (e.g., the first and second heterologous amino acid sequences are different), there may be two different species of regulator moiety. When there are two or more heterologous amino acid sequences that are the same species of heterologous amino acid sequence (e.g., the first and second heterologous amino acid sequences are the same), there is typically one species of regulator moiety. The regulator moiety of the first heterologous amino acid sequence may be the same or different from the regulator moiety of the second heterologous amino acid sequence, and preferably may be the same. The regulator moiety of the first heterologous amino acid sequence may be linked to the regulator moiety of the second heterologous amino acid sequence.

[0046] The regulator moiety can be any ligand, analyte, small organic molecule, ion, epitope, domain, fragment, subunit, moiety, or combination thereof. The regulator moiety can be a peptide or protein, including antibodies and antibody fragments, antigens, enzymes, phosphoproteins, glycoproteins, lipoproteins, and glycoproteins. The regulator moiety can be a lipid, phospholipid, carbohydrate (including simple sugars, disaccharides, and polysaccharides), nucleic acid, nucleoprotein, or any other molecule or analyte. The regulator moiety can be a small molecule, such as a drug or other pharmaceutical, including antibiotics. The regulator moiety can be anything that induces a conformational change in the heterologous amino acid sequence to activate the reporter protein, or that otherwise facilitates the interaction between the heterologous amino acid sequence and the reporter protein. Thus, the regulator moiety can be a modification, such as a covalently added post-translational modification (e.g., phosphorylation, glycosylation, sumoylation, lipidation, etc.), proteolytic cleavage (e.g., by a protease), or ligation (e.g., formation of a disulfide bond, for example, to generate a redox sensor). The modulator moiety can be a peptide or a small molecule.

[0047] Preferably, the regulator moiety is a peptide. Preferably, the first and second regulator moieties are peptides. The peptides may have any sequence, but are capable of binding to a heterologous amino acid sequence. Thus, the regulator moiety may comprise or consist of a peptide capable of binding to a heterologous amino acid sequence as defined herein. Typically, binding of the peptide to a heterologous amino acid sequence causes a conformational change or structural rearrangement of the heterologous amino acid sequence. Thus, when the regulator moiety is a peptide (i.e., a regulator peptide), the sequence of the peptide typically depends on the nature or identity of the heterologous amino acid sequence. Thus, when there are two or more different species of heterologous amino acid sequences (e.g., the first and second heterologous amino acid sequences are different), there may be two or more different species of regulator peptides (e.g., the first and second regulator moieties are different). When there are two or more heterologous amino acid sequences that are the same species or type of heterologous amino acid sequence (e.g., the first and second heterologous amino acid sequences are the same), there is typically one species or type of regulator peptide (e.g., the first and second regulator moieties are the same).

[0048] When at least one of the heterologous amino acid sequences is a calmodulin protein or a functional fragment thereof, at least one of the regulator moieties may be a calmodulin-binding peptide or a variant thereof. When all (i.e., the first and second, and optionally any further) heterologous amino acid sequences are each a calmodulin protein or a functional fragment thereof, the regulator moieties (i.e., the first and second regulator moieties, and optionally any further regulator moieties) are typically calmodulin-binding peptides or variants thereof. Thus, in a preferred example, the regulator moieties are calmodulin-binding peptides or variants thereof. In some examples, the first and second (and optionally any further) heterologous amino acid sequences are each a calmodulin protein or a variant or functional fragment thereof, and the first and second (and optionally any further) regulator moieties are each a calmodulin-binding peptide or a variant thereof, and the binding of the calmodulin-binding peptide to the calmodulin protein activates the activity of the reporter protein.

[0049] Calmodulin-binding peptides (CaM-BPs) preferably comprise any amino acid sequence (preferably a linear peptide epitope of 1-20 amino acids) that can specifically bind to calmodulin (i.e., a calmodulin protein as defined herein, or a variant or functional fragment thereof) and induce a structural rearrangement or conformational change in calmodulin that preferably brings the N-terminus and C-terminus of calmodulin into close proximity. Preferably, CaM-BPs can bind to a heterologous amino acid sequence, a calmodulin protein or a functional fragment thereof, provided as an insertion within the amino acid sequence of a reporter protein. Such binding typically reversibly modulates, and preferably activates, the activity of the reporter protein, as further described herein. A "CaM-BP" can be any previously described CaM-BP (see, e.g., Kursula, P., Amino Acids (2014), 46(10):2295-304) or variants thereof, including derivatives or variants of the specific CaM-BPs described herein. CaM-BP may comprise a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80% sequence identity, preferably at least 90% sequence identity, most preferably at least 95% sequence identity to any one of SEQ ID NOs: 17-35, preferably any one of SEQ ID NOs: 17, 18 or 19, most preferably at least 95% sequence identity to SEQ ID NO: 17. CaM-BP may comprise a contiguous sequence of at least 5, 6, 7, 8, 9 or 10, optionally at least 5, preferably at least 10, amino acids found in any one of SEQ ID NOs: 17-35, preferably any one of SEQ ID NOs: 17, 18 or 19, most preferably SEQ ID NO: 17, which is capable of binding to calmodulin. CaM-BP may comprise or consist of any one of SEQ ID NOs: 17-35, preferably any one of SEQ ID NOs: 17, 18 or 19, most preferably SEQ ID NO: 17, or a variant thereof.Preferably, CaM-BP may comprise or consist of any one of the sequences SEQ ID NO: 17, 18 or 19, most preferably SEQ ID NO: 17, or a variant thereof. The variant typically comprises any one of SEQ ID NO: 17-35, preferably any one of SEQ ID NO: 17, 18 or 19, most preferably a sequence having 1-5 amino acid deletions or insertions relative to the sequence SEQ ID NO: 17. The variant preferably comprises any one of SEQ ID NO: 17-35, preferably any one of SEQ ID NO: 17, 18 or 19, most preferably a sequence having 1-10 mutations, preferably 1-5 mutations relative to the sequence SEQ ID NO: 17. The variant retains binding to calmodulin. In some examples, CaM-BP may have a reduced binding affinity to calmodulin, i.e., CaM-BP may have a lower or reduced binding affinity to calmodulin protein compared to the binding affinity of wild-type CaM-BP to calmodulin protein. An exemplary wild-type CaM-BP is a peptide having the sequence SEQ ID NO: 17. An exemplary CaM-BP having increased binding affinity for calmodulin compared to wild-type CaM-BP may have the sequence of SEQ ID NO: 18. A CaM-BP having reduced binding affinity for calmodulin may preferably comprise or consist essentially of the sequence of SEQ ID NO: 19 or a variant thereof. An exemplary CaM-BP having reduced binding affinity for calmodulin compared to wild-type CaM-BP may have the sequence of SEQ ID NO: 19.

[0050] When at least one of the heterologous amino acid sequences is an affinity clamp or a functional fragment thereof, at least one of the regulator moieties can be an affinity clamp-binding peptide ligand (such as an RGS peptide ligand) or a variant thereof. When all (i.e., the first and second, and optionally any further) heterologous amino acid sequences are each an affinity clamp or a functional fragment thereof, the regulator moieties (i.e., the first and second regulator moieties, and optionally any further regulator moieties) are typically affinity clamp-binding peptide ligands (such as an RGS peptide ligand) or a variant thereof. Thus, the regulator moieties can be affinity clamp-binding peptide ligands (such as an RGS peptide ligand) or a variant thereof. In some examples, the first and second (and optionally any further) heterologous amino acid sequences are each an affinity clamp or a variant or functional fragment thereof, and the first and second (and optionally any further) regulatory moieties are each an affinity clamp binding peptide ligand (such as an RGS peptide ligand) or a variant thereof, and binding of the affinity clamp binding peptide ligand (such as the RGS peptide ligand) to the affinity clamp activates activity of the reporter protein.

[0051] The affinity clamp-binding peptide ligand (e.g., RGS peptide ligand) preferably comprises any amino acid sequence (preferably a linear peptide epitope of 1-20 amino acids) that is capable of specifically binding to an affinity clamp as defined herein and preferably capable of inducing a structural rearrangement or conformational change in the affinity clamp. Preferably, the affinity clamp-binding peptide ligand (such as an RGS peptide ligand) is capable of binding to a heterologous amino acid sequence that is an affinity clamp or a functional fragment thereof, provided as an insertion within the amino acid sequence of the reporter protein. Such binding typically reversibly modulates, and preferably activates, the activity of the reporter protein, as further described herein. An affinity clamp binding peptide ligand (such as an RGS peptide ligand) may comprise a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80% sequence identity, preferably at least 90% sequence identity, most preferably at least 95% sequence identity to SEQ ID NO: 82. An affinity clamp binding peptide ligand (such as an RGS peptide ligand) may comprise a contiguous sequence of at least 3, 4, 5, 6, 7, or 8, optionally at least 3, preferably at least 5, amino acids found in SEQ ID NO: 82 that can bind to an affinity clamp. An affinity clamp binding peptide ligand (such as an RGS peptide ligand) may comprise or consist of the sequence of SEQ ID NO: 82, or a variant thereof. Variants typically include sequences having a deletion or insertion of 1 to 5 amino acids, preferably 1 or 2 or 3 amino acids, most preferably 1 or 2 amino acids, relative to the sequence of SEQ ID NO: 82. Variants preferably include sequences containing 1 to 5 mutations, preferably 1 to 2 mutations, relative to the sequence of SEQ ID NO: 82. Variants retain binding to affinity clamps (e.g., SEQ ID NO: 76).

[0052] The regulator portion may be a peptide capable of binding to an affinity clamp (e.g., a PDZ domain-binding peptide), in which case at least one of the heterologous amino acid sequences may be an affinity clamp (e.g., an ePDZ domain). The regulator portion may be an SH3 domain-binding peptide, in which case at least one of the heterologous amino acid sequences may be an SH3 domain). The regulator portion may be an antibody-binding peptide or an antigen, in which case at least one of the heterologous amino acid sequences may be an antibody. The regulator portion may be a leucine zipper peptide, in which case at least one of the heterologous amino acid sequences may be a second leucine zipper peptide. The regulator portion may be a small molecule, solute, or peptide capable of binding to a solute-binding protein, as further described herein, in which case at least one of the heterologous amino acid sequences may be a solute-binding protein. Depending on the number of different species of heterologous amino acid sequences included in the reporter protein, there may be multiple different species of regulator portions. Thus, the modulator portion may be selected from a calmodulin-binding peptide or variant thereof; a peptide that binds to an affinity clamp; an SH3 domain-binding peptide; an antibody-binding peptide; a leucine zipper peptide; a small molecule, solute or peptide that binds to a solute-binding protein; or a combination thereof.

[0053] The peptide can be 1-10, 1-15, 1-20, 1-30, 1-40, 1-50, 1-100, 1-200 amino acids long, preferably 1-20 amino acids long. The peptide can comprise or consist of a linear binding epitope that binds to a heterologous amino acid sequence. In some examples, the regulator moiety is a peptide that binds to a calmodulin protein or a functional fragment thereof. In a preferred example, the first regulator moiety is a CaM-BP and the second regulator moiety is a CaM-BP. Typically, binding of the first CaM-BP to the first heterologous amino acid sequence, which is a calmodulin protein or a fragment thereof, and binding of the second CaM-BP to the second heterologous amino acid sequence, which is a calmodulin protein or a fragment thereof, activates the activity of the reporter protein.

[0054] The first and second regulator moieties can both be peptides, and the regulator moiety of the first heterologous amino acid sequence can have the same sequence or a different sequence (preferably the same sequence) as the regulator moiety of the second heterologous amino acid sequence. The first and second regulator moieties can both be peptides, and the first regulator moiety of the first heterologous amino acid sequence can be linked to the second regulator moiety of the second heterologous amino acid sequence, for example, via a linker (such as a peptide linker as further defined herein).

[0055] The regulator moiety may be a "caged" regulator moiety, e.g., the regulator moiety may be linked to a protein that can bind to the regulator moiety and thereby sequester the regulator moiety such that the regulator moiety is not free to bind to the heterologous amino acid sequence on the reporter protein. In such a case, the regulator moiety is preferably a peptide. The regulator moiety may be linked to a protein that corresponds to a variant of the heterologous amino acid sequence on the reporter protein but has a lower binding affinity for the regulator moiety compared to the heterologous amino acid sequence on the reporter protein. The regulator moiety may be linked to the protein via a linker, preferably a peptide linker. Thus, the caged regulator moiety may be a fusion protein comprising a peptide regulator moiety linked to a binding / sequestering protein via a peptide linker. The caged regulator moiety may preferably comprise a calmodulin binding peptide (CaM-BP) (e.g., any one of SEQ ID NOs: 17-35, preferably any one of SEQ ID NOs: 17, 18 or 19, most preferably SEQ ID NO: 17) linked (optionally via a peptide linker) to a calmodulin protein having reduced binding affinity for CaM-BP as described herein (such as SEQ ID NO: 16), which is typically the case when the first and second heterologous amino acid sequences on the reporter protein are calmodulin protein inserts. The first and second regulator moieties may both be caged regulator moieties, and optionally the first regulator moiety of the first heterologous amino acid sequence may be linked, for example via a linker, to the second regulator moiety of the second heterologous amino acid sequence.

[0056] In some instances, the peptide linker connecting the regulator portion and the binding / sequestering protein may comprise a protease cleavage site. The caged regulator portion may preferably comprise a calmodulin binding peptide (CaM-BP) (e.g., any one of SEQ ID NOs: 17-35, preferably any one of SEQ ID NOs: 17, 18 or 19, most preferably SEQ ID NO: 17) linked via a peptide linker comprising a protease cleavage site to a calmodulin protein having reduced binding affinity for CaM-BP, as described herein (e.g., SEQ ID NO: 16), typically when the first and second heterologous amino acid sequences on the reporter protein are calmodulin protein insertions. The caged regulator portion may comprise two CaM-BPs and two calmodulin proteins having reduced binding affinity for CaM-BPs, the two calmodulin proteins linked via peptide linkers, each calmodulin protein linked to a respective CaM-BP via a peptide linker, each linker comprising a protease cleavage site. The caged regulator portion may comprise one CaM-BP and two calmodulin proteins with reduced binding affinity to the CaM-BP, the two calmodulin proteins linked via a peptide linker, one of the calmodulin proteins linked to the CaM-BP via a peptide linker, each linker comprising a protease cleavage site. The caged regulator portion may comprise one CaM-BP, two calmodulin proteins with reduced binding affinity to the CaM-BP, and a protease binding domain that specifically binds to a protease; the two calmodulin proteins linked via a peptide linker comprising a target cleavage site of the protease, one of the calmodulin proteins linked to the CaM-BP via a peptide linker comprising a target cleavage site of the protease, the other calmodulin protein linked to the protease binding domain, and optionally the protease binding domain bound to the protease. Such caged regulator portion reduces background activation of the reporter protein / biosensor.Such caged regulatory moieties containing a protease cleavage site can be used in biosensors to detect proteases, where the protease cleavage site is the target cleavage site of a protease of interest.

[0057] joining part The reporter proteins described herein are typically configured to be activated in the presence of one or more target molecules (i.e., one or more species of target molecules). Thus, the reporter protein further comprises at least one, preferably two binding moieties. The reporter protein may comprise a binding moiety (B1'). The reporter protein may comprise a first binding moiety (B1') and a second binding moiety (B2'). The binding moiety on the reporter protein typically interacts and / or binds to a target molecule of the biosensor. Typically, each regulator moiety comprises at least one, preferably one binding moiety. The binding moiety on the regulator moiety may interact and / or bind to a target molecule of the biosensor. Each binding moiety of the reporter protein typically forms an interaction pair with a respective binding moiety on the regulator moiety. Thus, each regulator moiety may comprise a binding moiety capable of interacting with at least one of the binding moieties on the reporter protein. The regulator moiety may comprise a binding moiety (B1'') capable of interacting with the binding moiety (B1') on the reporter protein. The first regulator portion may comprise a binding moiety (B1″) capable of interacting with a first binding moiety (B1′) on the reporter protein, and the second regulator portion may comprise a binding moiety (B2″) capable of interacting with a second binding moiety (B2′) on the reporter protein. Preferably, the binding moiety (B1″) on the first regulator portion forms an interaction pair with the first binding moiety (B1′) on the reporter protein, and the binding moiety (B2″) on the second regulator portion forms an interaction pair with the second binding moiety (B2′) on the reporter protein. The first regulator portion and the second regulator portion may be linked (e.g., optionally via a peptide linker as a fusion protein) and may further comprise a binding moiety (B1″) capable of interacting with the binding moiety (B1′) on the reporter protein.

[0058] Each pair of binding moieties may directly interact with each other, i.e. bind directly. Preferably, each pair of binding moieties interacts via binding to a target molecule. Thus, the interaction between the binding moiety on the reporter protein and the binding moiety on the regulator moiety may depend on the presence of the target molecule. Each pair of binding moieties may bind to the same target molecule at the same time (i.e. simultaneously). The binding moiety (B1') on the reporter protein and the binding moiety (B1'') on the regulator moiety may both bind to the target molecule (TM1) simultaneously. Alternatively, the binding moiety (B1') on the reporter protein may bind to the target molecule (TM1) and the binding moiety (B1'') on the regulator moiety may bind to a complex of B1' and TM1; or the binding moiety (B1'') on the regulator moiety may bind to the target molecule (TM1) and the binding moiety (B1') on the reporter protein may bind to a complex of B1'' and TM1. The first binding moiety (B1') on the reporter protein and the binding moiety (B1'') on the first regulator moiety can both simultaneously bind to a first target molecule (TM1); the second binding moiety (B2') on the reporter protein and the binding moiety (B2'') on the second regulator moiety can both simultaneously bind to a second target molecule (TM2). The first binding moiety (B1') on the reporter protein may bind to a first target molecule (TM1) and the binding moiety (B1'') on the first regulator portion may bind to a complex of B1' and TM1, or the binding moiety (B1'') on the first regulator portion may bind to a first target molecule (TM1) and the first binding moiety (B1') on the reporter protein may bind to a complex of B1' and TM1; the second binding moiety (B2') on the reporter protein may bind to a second target molecule (TM2) and the binding moiety (B2'') on the second regulator portion may bind to a complex of B2' and TM2, or the binding moiety (B2'') on the second regulator portion may bind to a second target molecule (TM2) and the second binding moiety (B2') on the reporter protein may bind to a complex of B2' and TM2. The interaction of binding moieties B1' and B1" may depend on the presence of a first target molecule TM1, and the interaction of binding moieties B2' and B2" may depend on the presence of a second target molecule TM2. The reporter protein may comprise further binding moieties (BX'; e.g., B3', B4', etc.).The reporter protein may contain as many binding moieties as there are heterologous amino acid sequences contained in the reporter protein, and each such additional binding moiety forms an interaction pair with a respective binding moiety (e.g., BX' interacts with BX"), and each binding moiety (BX") is contained on an additional regulator moiety.

[0059] The binding moieties on the reporter protein may be of the same or different species (i.e., the reporter protein may only contain one species of binding moiety, or it may contain two different species of binding moieties). The binding moieties on the regulator moiety may be of the same or different species (i.e., each regulatory moiety may contain the same (i.e., a single type) binding moiety, or there may be many different species of regulatory moieties, each species of regulatory moiety containing a different binding moiety, preferably there are two different species of regulatory moieties). Each binding moiety pair may interact with the same species of target molecule, such that one species (i.e., one type) of target molecule regulates the activity of the reporter protein. Alternatively, each binding moiety pair may interact with different species of target molecule, such that two or more species (i.e., types) of target molecule regulate the activity of the reporter protein. A species of target molecule may be considered to refer to multiple target molecules that are all the same, i.e., the same molecule, compound or protein. Thus, the binding moieties B1' and B2' on the reporter protein may be the same species of binding moiety, in which case typically the respective binding moieties B1" and B2" on the regulator moiety are the same species of binding moiety. In such cases, TM1 and TM2 are usually the same species of target molecule. Alternatively, the binding moieties B1' and B2' on the reporter protein may be different species of binding moieties, in which case typically the respective binding moieties B1" and B2" on the regulator moiety are different species of binding moieties. In such cases, TM1 and TM2 are usually different species of target molecule.

[0060] The reporter protein may comprise a first heterologous amino acid sequence responsive to binding of a first regulator portion and a second heterologous amino acid sequence responsive to binding of a second regulator portion, where binding of the first regulator portion to the first heterologous amino acid sequence and binding of the second regulator portion to the second heterologous amino acid sequence reversibly regulates the activity of the reporter protein; the reporter protein may further comprise a first binding moiety B1' and a second binding moiety B2'. The first regulator portion may further comprise a binding moiety B1" and the second regulator portion may further comprise a binding moiety B2". The binding moiety B1" of the first regulator portion may be capable of interacting with the first binding moiety B1' of the reporter protein and the binding moiety B2" of the second regulator portion may be capable of interacting with the second binding moiety B2' on the enzyme. Preferably, the interaction of binding moieties B1' and B1" depends on the presence of a first target molecule TM1, and the interaction of binding moieties B2' and B2" depends on the presence of a second target molecule TM2. Thus, binding moieties B1' and B1" may form a binding moiety pair that simultaneously binds to target molecule TM1, and binding moieties B2' and B2" may form a binding moiety pair that simultaneously binds to target molecule TM2. Binding moieties B1' and B1" and binding moieties B2' and B2" may be the same binding moiety pair or different binding moiety pairs. Target molecules TM1 and TM2 may be the same or different types of target molecules.

[0061] In some examples, the reporter protein comprises a binding moiety B1' capable of interacting with a respective binding moiety B1" typically via the target molecule TM1. Each binding moiety B1" may be linked (optionally via a peptide linker) to a first regulator moiety and a second regulator moiety, which are configured to bind to a first and a second heterologous amino acid sequence, respectively, on the reporter protein, thereby activating the reporter protein in the presence of TM1. In some preferred examples, the reporter protein comprises two calmodulin protein inserts and a binding moiety B1', each binding moiety B1" being linked (preferably via a peptide linker, optionally as a fusion protein) to two CaM-BPs, preferably CaM-BPs having reduced binding affinity for calmodulin compared to wild-type CaM-BP. The reporter protein may comprise two calmodulin protein inserts and a binding moiety B1', each binding moiety B1'' linked (preferably via a peptide linker, optionally as a fusion protein) to two caged CaM-BPs, as further described herein, each caged CaM-BP preferably comprising a CaM-BP linked to a calmodulin protein that has reduced binding affinity for the CaM-BP compared to the calmodulin protein insert on the reporter protein.

[0062] As generally used herein, a "binding moiety" or "binding moieties" refers to one or more molecules or biological or chemical components or entities that can recognize, interact, and / or bind to each other, or preferably to one or more target molecules. The exact identity of the binding moieties used in the biosensors and reporter proteins described herein typically depends on the target molecule or molecules to be detected by the reporter protein / biosensor. Thus, the binding moieties of the reporter protein / biosensor can be selected to bind to the target molecule(s) of interest. The binding moieties can be polypeptides, nucleic acids (e.g., single- or double-stranded DNA or RNA), sugars, oligosaccharides, polysaccharides or other carbohydrates, lipids, or any combination of these, such as glycoproteins, PNA constructs, or molecular components thereof.By way of example only, binding moieties can include (i) an amino acid sequence of a ligand binding domain of a receptor responsive to binding of a target molecule, such as a cognate growth factor, a cytokine, a hormone (e.g., insulin), or a neurotransmitter; (ii) an amino acid sequence of an ion or metabolite transporter capable of binding or responsive to a target molecule, such as an ion or metabolite (e.g., a Ca2+ binding protein, such as calmodulin or calcineurin, or a glucose transporter, or a solute binding protein); (iii) a zinc finger amino acid sequence responsive to zinc-dependent binding of a DNA target molecule; (iv) a helix-loop-helix amino acid sequence responsive to binding of a DNA target molecule; (v) a pleckstrin homology domain amino acid sequence responsive to binding of a phosphoinositide target molecule; (vi) a Src signaling protein responsive to a phosphoinositide target molecule; (vii) an amino acid sequence of an antigen responsive to binding of an antibody target molecule; or (viii) an amino acid sequence of a protein kinase or phosphatase responsive to binding of a phosphorylatable or phosphorylated target molecule; (ix) a ubiquitin-binding domain; (x) a protein or protein domain that binds a small molecule, drug or antibiotic, such as rapamycin-binding FKBP and FRB domains; (xi) a single- or double-stranded DNA, RNA or PNA construct that binds a nucleic acid target molecule, e.g., where DNA or RNA is coupled or crosslinked to an amino acid sequence or other protein-nucleic acid interactions; and / or (xii) an affinity clamp, such as a PDZ-FH3 domain fusion; including modified or engineered versions thereof. In some cases, one of the binding moieties may be a small molecule binding protein, and each binding moiety may recognize (i.e., specifically bind) a "small molecule:binding moiety" complex, but not recognize the small molecule or binding moiety alone. For example, binding moiety B1' or B2' on the reporter protein may bind to small molecule X and binding moiety B1" or B2" may bind to the X:B1' or X:B2' complex, respectively, or vice versa. Preferably, the binding moieties are proteins. Preferably, the binding moiety pair are proteins that can interact with each other directly or, preferably, through binding to a target molecule.

[0063] Exemplary binding moieties are given in the list below, some of which are described in the examples. Thus, the binding moieties (e.g., B1' and B1'' and / or B2' and B2''') may be selected from the group consisting of FKBP (SEQ ID NO: 36), FRB (SEQ ID NO: 37), calcineurin α and β subunits (including SEQ ID NO: 38 and 39 or including SEQ ID NO: 40), human serum albumin (HAS) GA binder (SEQ ID NO: 41), cyclophilin (SEQ ID NO: 42), antibody fragments, in particular α-amylase binding antibody VHH fragments, VHH1 (SEQ ID NO: 43) and VHH2 (SEQ ID NO: 44), HAS antibody VHH binder (SEQ ID NO: 45), methotrexate binding antibody VHH fragment (SEQ ID NO: 46) or nanoCLAMP for methotrexate binding (SEQ ID NO: 47); and variants thereof. The skilled artisan can easily select compatible binding moiety pairs that are preferably capable of interacting via simultaneous binding to the target molecule. The variants are typically functional binding variants of the respective binding moieties involved. One skilled in the art can generate antibodies and antibody (VHH) fragments targeting any number of known target molecules for use as binding moieties in the reporter proteins / biosensors described herein. It will also be understood that the binding moieties may be modified or chemically derivatized with binding agents such as biotin, avidin, epitope tags, lectins, carbohydrates or lipids. As will be readily understood by one skilled in the art, suitable binding moieties can be designed to bind any target molecule of interest, for example, by generating a VHH-single domain antibody (VHH1) that binds to a target molecule (TMX), which may preferably be a peptide or small molecule, and then generating a second VHH-single domain antibody (VHH2) to recognize the "TMX:VHH1" complex. It is routine for one skilled in the art to generate such VHH single domain antibodies with the required specificity for a known target.

[0064] The binding moieties may be or may comprise antibodies or antibody fragments, including monoclonal and polyclonal antibodies, recombinant antibodies, Fab and Fab'2 fragments, DARPins, diabodies, monobodies, nanoCLAMPs (CLostridal Antibody Mimetic Proteins, see e.g. SEQ ID NO: 47), and single chain antibody fragments (e.g. scV). Suitably, the first and second binding moieties may be or may comprise respective antibodies or antibody fragments (preferably scFv) that bind to the target molecule.

[0065] In some examples, the binding moieties (e.g., B1' and B1'' and / or B2' and B2''') are or include the amino acid sequence of an affinity clamp. An affinity clamp preferably includes a recognition domain and optionally an enhancer domain. The recognition domain is typically capable of binding to one or more target molecules, such as those described in (i)-(ix) above. Recognition domains include domains involved in phospho-tyrosine binding (e.g., SH2, PTB), domains involved in phospho-serine binding (e.g., UIM, GAT, CUE, BTB / POZ, VHS, UBA, RING, HECT, WW, 14-3-3, Polo-box), domains involved in phospho-threonine binding (e.g., FHA, WW, Polo-box), domains involved in proline-rich region binding (e.g., EVH1, SH3, GYF), domains involved in acetylated lysine binding (e.g., Bromo), domains involved in methylated lysine binding (e.g., Chromo, PHD), domains involved in apoptosis (e.g., BIR, TRAF, DED, Death, CARD, BH), domains involved in cytoskeleton regulation (e.g., ADF, GEL, DH, CH, FH2), ubiquitin binding domains or modified or engineered versions thereof, or domains involved in other cellular functions (e.g., EH, CC, VHL, TUDOR, PUF). Enhancer domains may include, but are not limited to, the following: Repeat, PAS, MH1, LRR1, IQ, HEAT, GRIP, TUBBY, SNARE, TPR, TIR, START, SOCS Box, SAM, RGS, PDZ, PB1, LIM, F-BOX, ENTH, EF-Hand, SHADOW, ARM, ANK. Enhancer domains typically increase or enhance the binding affinity to at least one or target molecules. In some embodiments, the affinity may be increased by at least 10-fold, 100-fold, or 1000-fold compared to the affinity of the recognition domain alone. The affinity clamp may further comprise a linker connecting the recognition domain and the enhancer domain.

[0066] In some examples, the affinity clamp comprises a recognition domain comprising at least a portion or fragment of a PDZ domain and an enhancer domain comprising at least a portion or fragment of a fibronectin type III domain. The PDZ domain may be derived from human Erbin protein. Erbin-PDZ (ePDZ) binds to a target molecule such as the C-terminus of p120-associated catenin (such as delta-catenin and the armadillo repeat gene deleted in palatocardiofacial syndrome (ARVCF)). Preferably, the affinity clamp in this case further comprises the tenth (10th) type III (FN3) domain of human fibronectin as an enhancer domain. In some examples, the affinity clamp may comprise one or more connector amino acid sequences. For example, the connector amino acid sequence may connect a protease amino acid sequence (e.g., comprising a protease amino acid sequence) to the Erbin-PDZ domain, the Erbin-PDZ domain to the FN3 domain, and / or the FN3 domain to an inhibitor. For a more detailed description of the structure and function of affinity clamps, and of particular affinity clamps that can be used according to the invention, see also WO2009 / 062170, Zhuang & Liu, 2011, Comput. Theoret. Chem. 963 448, Huang et al, 2009, J. Mol. Biol. 392 1221, Huang et al, 2008, PNAS (USA) 105 6578, and Koide and Huang Methods Enzymol. 2013; 523: 285-302. Preferably, the affinity clamp may comprise or consist of the sequence of SEQ ID NO: 76 (e.g., as a recognition domain), or a variant or functional fragment thereof, and may bind to an RGS peptide ligand comprising or consisting of the sequence of SEQ ID NO: 82, or a variant thereof. Thus, each of the binding moieties B1' or B2' may optionally comprise or consist of an affinity clamp comprising or consisting of the sequence of SEQ ID NO: 76, and each of the binding moieties B1'' or B2'' may optionally comprise or consist of an RGS peptide ligand comprising or consisting of the sequence of SEQ ID NO: 82, or vice versa.

[0067] Each binding moiety in each binding moiety pair (i.e., the binding moiety on the reporter protein (e.g., B1' or B2') and the binding moiety on the regulatory moiety (e.g., B1" or B2")) can directly interact. Preferably, each binding moiety in each binding moiety pair (i.e., the binding moiety on the reporter protein (e.g., B1' or B2') and the binding moiety on the regulatory moiety (e.g., B1" or B2")) can bind, interact, or form a complex with a target molecule (e.g., TM1 or TM2). A target molecule may comprise multiple different subunits, domains, or epitopes, each of which may be bound by a respective binding moiety in the pair, thereby colocalizing the binding moiety pair and thus further components of the biosensor / reporter protein described herein. Thus, a direct binding interaction between a target molecule and a binding moiety pair suitably promotes colocalization of components of the biosensor described herein.

[0068] The binding moiety may be linked (i.e. conjugated, joined or fused) to the N-terminus or C-terminus of the reporter protein, optionally via a linker. The reporter protein may comprise one binding moiety B1', which may be linked to the N-terminus or C-terminus of the reporter protein, optionally via a linker. The reporter protein may comprise two binding moieties B1' and B2', where the first binding moiety (B1') may be linked to the N-terminus of the reporter protein, optionally via a linker, and the second binding moiety (B2') may be linked to the C-terminus of the reporter protein, optionally via a linker; or vice versa. The reporter protein and the one or more binding moieties (e.g. the first and second binding moieties B1' and B2') may preferably be provided as a fusion protein. The reporter protein and the one or more binding moieties may be provided as a single contiguous amino acid sequence. A single continuous amino acid sequence may be provided that includes, from N-terminus to C-terminus or C-terminus to N-terminus, the amino acid sequence of the binding moiety (B1'), optionally a linker, and the amino acid sequence of the reporter protein comprising the first and second heterologous amino acid sequences. A single continuous amino acid sequence may be provided that includes, from N-terminus to C-terminus, the amino acid sequence of the first binding moiety (B1'), optionally a linker, the amino acid sequence of the reporter protein comprising the first and second heterologous amino acid sequences, optionally a linker, and the amino acid sequence of the second binding moiety (B2'). The amino acid sequences of the first and second binding moieties may be the same or different. The binding moieties (e.g., B1' and / or B2') may be inserted into the amino acid sequence of the reporter protein, optionally adjacent to the linker. Typically, the binding moieties (e.g., B1' and / or B2') may be inserted into the amino acid sequence of the reporter protein at a position that does not affect or does not affect the activity of the reporter protein, for example in a loop region. Suitable linkers are as described elsewhere herein.

[0069] A binding moiety (e.g., B1″ or B2″) may be linked (i.e., conjugated or fused) to a regulator moiety. A first binding moiety (B1″) may be linked to a first regulator moiety and a second binding moiety (B2″) may be linked to a second regulator moiety. When a reporter protein comprises the same first and second heterologous amino acid sequences and comprises the same first binding moiety (B1′) and second binding moiety (B2′) (B1′=B2′), a single species of regulator moiety may be linked to a binding moiety that can interact with both the first and second binding moieties. When a reporter protein comprises one binding moiety (B1′), each binding moiety B1″ may be linked to both the first and second regulator moieties. When a regulatory moiety is a protein or peptide, a binding moiety (e.g., B1″ or B2″) may be linked to the N-terminus or C-terminus of the regulatory moiety, optionally via a linker. Each regulator moiety may comprise one binding moiety. The regulator portion and the binding portion may be provided as a fusion protein. The regulator portion and the binding portion may be provided as a single contiguous amino acid sequence. From N-terminus to C-terminus: the amino acid sequence of the binding portion, optionally a linker, and the amino acid sequence of the regulatory portion; or from N-terminus to C-terminus, a single contiguous amino acid sequence may be provided that includes the amino acid sequence of the regulatory portion, optionally a linker, and the amino acid sequence of the binding portion (e.g., B1″-RM1, RM1-B1″, B2″-RM2, RM2-B2″, “-” may include a linker). The binding portion may be inserted within the amino acid sequence of the regulatory portion, and optionally the amino acid sequence of the binding portion may be adjacent to the linker. Typically, in such cases, the binding portion may be inserted within the amino acid sequence of the regulatory portion at a position that does not affect or does not affect the binding of the regulatory portion to the respective heterologous amino acid sequence. One binding portion may include two regulator portions. The binding portion and the two regulator portions may be provided as a fusion protein. The binding moiety and the two regulator moieties may be provided as a single contiguous amino acid sequence. A single contiguous amino acid sequence may be provided that comprises, from N-terminus to C-terminus, the first regulator moiety, optionally a linker, the binding moiety, optionally a linker, and the second regulator moiety.Suitable linkers are as described elsewhere herein.

[0070] target molecule The reporter proteins / biosensors described herein may be capable of detecting one or more target molecules. For each target molecule to be detected, the biosensor typically includes one or more binding moiety pairs capable of binding (usually at the same time) to that target molecule, and for each binding moiety pair, the reporter protein includes one binding moiety and the regulatory moiety includes a respective second binding moiety of the pair. The reporter proteins / biosensors may be capable of detecting one species or type of target molecule. The reporter proteins / biosensors may be capable of detecting two species or types of target molecules. The reporter proteins may include a first binding moiety B1' and a second binding moiety B2', and there may be two species of regulator moieties, the first regulator moiety includes a first binding moiety B1" and the second regulator moiety includes a second binding moiety B2", where the binding moieties B1' and B1" form a binding pair and can simultaneously interact with the target molecule TM1, and the binding moieties B2' and B2" form a binding pair and can simultaneously interact with the target molecule TM2. The target molecules TM1 and TM2 may be the same or different types of target molecules. The reporter protein may comprise a first binding moiety B1' and a second binding moiety B2' that are the same (B1'=B2'), and there may be one type of regulator moiety that comprises a binding moiety that forms a binding pair with either B1' or B2' in the presence of the target molecule TM1. The reporter protein may comprise one binding moiety B1', and the binding moiety B1'' that forms a binding pair with B1' in the presence of the target molecule TM1 is linked to a first and a second regulator moiety, which may be the same or different depending on the heterologous amino acid sequence of the reporter protein.

[0071] The target molecule (e.g., TM1 and / or TM2) can be any ligand, analyte, ion (e.g., calcium, Ca 2+), small organic molecules, epitopes, domains, fragments, subunits, portions, or combinations thereof. Target molecules (e.g., TM1 and / or TM2) can be, for example, antibodies and antibody fragments, antigens, enzymes such as α-amylase, proteins including human, serum albumin, phosphoproteins, glycoproteins, lipoproteins, and glycoproteins. Target molecules (e.g., TM1 and / or TM2) can be lipids, phospholipids, carbohydrates including simple sugars, disaccharides, and polysaccharides; nucleic acids, nucleoproteins. The target molecule (e.g., TM1 and / or TM2) may be a small molecule, chemical entity or any other analyte, including drugs such as immunosuppressants including rapamycin (i.e., sirolimus), cyclosporine and tacrolimus (i.e., FK506), as well as other pharmaceuticals including antibiotics, vitamins, banned substances, illegal drugs or drugs of addiction, chemotherapeutic agents and lead compounds in drug design and screening, molecules and analytes typically found in biological samples, such as biomarkers, tumor and other antigens, receptors, DNA binding proteins including transcription factors, hormones, neurotransmitters, growth factors, cytokines, receptors, metabolic enzymes, signaling molecules, nucleic acids such as DNA and RNA, membrane lipids and other cellular components, pathogen-derived molecules including viral, bacterial, protozoan, fungal and insect proteins, lipids, carbohydrates and nucleic acids. The target molecule may be a protease. This is particularly the case when the biosensor includes a caged regulator moiety as further described herein. Typically, in such cases, in the presence of the target protease of the biosensor, the protease cleaves the target protease cleavage site of the caged regulator portion, thereby releasing the regulator portion, which then binds to the heterologous amino acid sequence of the reporter protein, thereby activating the reporter protein. When the target molecule is a protease and the biosensor includes a caged regulator portion, the reporter protein may not include a binding domain. When the target molecule is a protease and the biosensor includes a caged regulator portion, the caged regulator portion may include a protease binding domain that can act to increase the local concentration of the target protease at the protease cleavage site of the caged regulator portion.

[0072] Preferably, the target molecule (e.g., TM1 and / or TM2) may be selected from the group consisting of (i) hormones including: fertility hormones, such as progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH); stress hormones (e.g., hormones related to psychological stress), such as cortisol and alpha-amylase; metabolic hormones (e.g., hormones related to or providing an indication of metabolic status), such as insulin and glucose; (ii) therapeutic agents such as methotrexate, rapamycin, tacrolimus, and cyclosporine A; and (iii) environmental pollutants, such as phenol glucosides. The reporter protein / biosensor may be configured to detect any one of the target molecules. The reporter protein / biosensor may be configured to detect any two of the target molecules. In a particularly preferred example, the target molecule (e.g., TM1 and / or TM2) may be methotrexate, phenol glucoside, rapamycin, tacrolimus, and / or cyclosporine A. The reporter protein / biosensor may be configured to detect any one of methotrexate, phenol glucoside, rapamycin, tacrolimus, and / or cyclosporine A. For example, TM1 and TM2 are the same and are selected from the group consisting of methotrexate, phenol glucoside, rapamycin, tacrolimus, and / or cyclosporine A. The reporter protein / biosensor may be configured to detect any two of methotrexate, phenol glucoside, rapamycin, tacrolimus, and / or cyclosporine A. For example, TM1 and TM2 are different and each independently are selected from the group consisting of methotrexate, phenol glucoside, rapamycin, tacrolimus, and / or cyclosporine A. The reporter protein / biosensor may be configured to detect rapamycin and cyclosporine A. For example, TM1 is rapamycin and TM2 is cyclosporine A. For example, TM1 is rapamycin and TM2 is rapamycin. For example, TM1 is cyclosporine A and TM2 is cyclosporine A.One skilled in the art will be readily able to select appropriate binding moiety pairs, typically proteins or antibodies, capable of binding to the selected target molecule(s) of interest using routine interaction screening and binding assays, such as, for example, ELISA or surface plasmon resonance methods such as Biacore.

[0073] Exemplary binding moieties and target molecules are described in the Examples. In some examples, the target molecule (e.g., TM1 and TM2) is an enzyme, such as alpha amylase. In such cases, the first and second binding moieties can be antibodies therefor, such as the exemplified camelid antibodies VHH1 and VHH2 (SEQ ID NOs: 43 and 44) ​​or variants thereof (e.g., B1' and B2' each comprise a VHH1 comprising the sequence of SEQ ID NO: 43; B1" and B2" each comprise a VHH2 comprising the sequence of SEQ ID NO: 44). In some examples, the target molecule (e.g., TM1 and TM2) is a small organic molecule, such as rapamycin. In such cases, the first and second binding moieties can be FKBP and FRB (SEQ ID NOs: 36 and 37), respectively, or variants thereof (e.g., B1' and B2' each comprise a FKBP comprising the sequence of SEQ ID NO: 36; B1" and B2" each comprise a FRB comprising the sequence of SEQ ID NO: 37). In some examples, the target molecule (e.g., TM1 and TM2) is a small organic molecule such as FK506 (i.e., tacrolimus). In such cases, the first and second binding moieties can be FKBP and calcineurin α / β complex (SEQ ID NO: 36, and 38 and 39 or 40), respectively, or variants thereof (e.g., B1' and B2' each comprise FKBP comprising the sequence of SEQ ID NO: 36; B1'' and B2'' each comprise calcineurin α / β complex comprising the sequence of SEQ ID NO: 38 and 39, or 40). In some examples, the target molecule (e.g., TM1 and TM2) is human serum albumin (HAS). In such cases, the first and second binding moieties can be a HAS GA binder and a HAS-specific VHH (SEQ ID NOs: 41 and 45), respectively, or variants thereof (e.g., B1' and B2' each comprise a HAS GA binder comprising the sequence of SEQ ID NO: 41; B1" and B2" each comprise a HAS-specific VHH comprising the sequence of SEQ ID NO: 45). In some examples, the target molecule is a cyclosporine (e.g., TM1 and TM2).In such a case, the first and second binding moieties can be cyclophilin (SEQ ID NO: 42) and calcineurin α / β complex (SEQ ID NO: 38 and 39, or 40), respectively, or variants thereof (e.g., B1' and B2' each comprise a cyclophilin comprising the sequence of SEQ ID NO: 38; B1" and B2" each comprise a calcineurin α / β complex comprising the sequence of SEQ ID NO: 38 and 39, or 40). In some examples, the target molecule (e.g., TM1 and TM2) is methotrexate (MTX). In such a case, the first and second binding moieties can be MTX-specific VHH and MTX-specific nanoCLAMP (SEQ ID NO: 46 and 47), respectively, or variants thereof (e.g., B1' and B2' each comprise a MTX-specific VHH comprising the sequence of SEQ ID NO: 46; B1" and B2" each comprise a MTX-specific nanoCLAMP comprising the sequence of SEQ ID NO: 47). In some examples, the target molecules are rapamycin and cyclosporine (e.g., TM1 is rapamycin and TM2 is cyclosporine). In such cases, binding moiety B1' can include FKBP (SEQ ID NO: 36), binding moiety B2' can include cyclophilin (SEQ ID NO: 42), binding moiety B1" can include FRB (SEQ ID NO: 37), and binding moiety B2" can include calcineurin α / β complex (SEQ ID NOs: 38 and 39, or 40).

[0074] Biosensors The reporter proteins described herein are designed to be incorporated into biosensors, for example, for the detection of target molecules. Biosensors comprising the reporter proteins described herein are described herein. The biosensors typically comprise (i) a reporter protein described herein; and (ii) one or more regulatory moieties described herein. The biosensors preferably comprise (i) a reporter protein described herein; (ii) a first regulator moiety described herein; and (iii) a second regulator moiety described herein. The biosensors may comprise (i) a reporter protein described herein; and (ii) one or more caged regulator moieties described herein. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme (i.e., an enzyme substrate or substrate molecule), as further described herein.

[0075] The function of the reporter protein / biosensor described herein is based on the principle that a reporter protein can have a series of conformations in which it is inactive. Although the conformation of an active reporter protein is likely to be similar to that of the wild-type parent molecule, there may be several different conformations in which the reporter protein is inactive. Thus, several conformation-induced inactivation changes in the reporter protein can be induced by two or more insertions of heterologous amino acid sequences at different insertion positions. The conformational changes at each of these insertion sites can then epistatically interact or synergistically bind to convert the reporter protein from an inactive state to an active state.

[0076] Thus, typically, the binding of the regulator moiety to the heterologous amino acid sequence induces a conformational change in the heterologous amino acid sequence, which induces a conformational change in the reporter protein. The binding of the regulator moiety at each of the heterologous amino acid sequences contained in the reporter protein (e.g., the binding of the first regulator moiety to the first heterologous amino acid sequence and the binding of the second regulator moiety to the second heterologous amino acid sequence) induces several conformational changes that together induce a conformational change in the reporter protein that converts it from an inactive state to an active state. Thus, typically, only the binding of the regulator moiety at all of the heterologous amino acid sequences contained in the reporter protein activates the reporter protein. Thus, the reporter protein / biosensor of the present invention may be characterized as an "AND-gate" biosensor, since two simultaneous inputs (e.g., the first and second regulator moieties binding to the first and second heterologous amino acid sequences, respectively) are required to trigger the activity of the reporter protein to produce an output, i.e., a detectable and / or measurable signal.

[0077] The activity of the reporter protein may ultimately depend on the presence of one or more target molecules (e.g., TM1, or TM1 and TM2). Typically, the reporter protein / biosensor of the present invention is designed to detect one or two target molecules (e.g., TM1=TM2, or TM1 and TM2). Typically, the reporter protein comprises one half of a binding moiety pair (e.g., B1' or B2'), and the other half of each of the binding moiety pairs (e.g., B1" or B2") is comprised on a regulatory moiety. Typically, the binding moiety pair (e.g., B1' and B1", or B2' and B2") interacts through mutual recognition of the target molecule (e.g., TM1 or TM2, respectively). Thus, in the presence of the target molecule, the binding moiety pair binds to the target molecule simultaneously, thereby co-localizing the regulatory moiety with the reporter protein. This enhances or promotes the binding of the regulatory moiety to the heterologous amino acid sequence comprised in the reporter protein. There may be two binding moiety pairs in the biosensor, such that the reporter protein comprises two binding moieties, each forming one half of two binding moiety pairs, where the other half of each of the pairs is located on the regulatory moiety.

[0078] For detection of one target molecule, the two binding moiety pairs can be the same, so that in the presence of the target molecule, both binding moiety pairs bind simultaneously to the respective target molecule, colocalizing the reporter protein with the two regulatory moieties. Alternatively, for detection of one target molecule, the reporter protein can comprise one binding moiety, and each second binding moiety can be linked to two regulator moieties, so that in the presence of the target molecule, a single binding moiety pair binds to the target molecule, colocalizing the reporter protein with the two regulatory moieties. In either case, this enhances and promotes the binding of the regulator moiety to each of the heterologous amino acid sequences contained in the reporter protein, thus inducing two sets of conformational changes in the reporter protein, thereby converting it from an inactive state to an active state. The need for two separate inputs results in low background noise (e.g., because random activation of the reporter protein is highly unlikely) and a high dynamic range of the reporter protein. It also appears to favor a higher maximum catalytic activity of the biosensor compared to single insertion variants. Typically, there is a trade-off between dynamic range and catalytic activity. Surprisingly, it has been found that by using two high catalytic activity and low dynamic range modules, it is possible to avoid this trade-off and generate a module with both high catalytic activity and high dynamic range.This is exemplified by the biosensor described herein, such as the BLA-2CaM MTX biosensor.To require additional input, additional binding moiety pairs and additional heterologous amino acid sequences can be included, which can further improve the dynamic range of reporter protein.

[0079] To detect two target molecules, the two binding moiety pairs may be different, so that in the presence of only one of the target molecules, only one binding moiety pair will simultaneously bind to the target molecule, colocalizing the reporter protein with only a single regulatory moiety. The presence of a second target molecule is necessary for the activation of the reporter protein, so that in the presence of the first and second target molecules, both binding moiety pairs will simultaneously bind to the respective target molecules, colocalizing the reporter protein with the two regulatory moieties. The regulatory moiety binds to each of the heterologous amino acid sequences contained in the reporter protein, thus inducing two sets of conformational changes in the reporter protein, thereby converting it from an inactive state to an active state. Thus, the need for two separate inputs can be used to detect the simultaneous presence of two target molecules.

[0080] Thus, in some examples, in the presence of a first target molecule (TM1), a first binding moiety (B1') on the reporter protein interacts with the respective binding moiety (B1") on the first regulator moiety; in the presence of a second target molecule (TM2), a second binding moiety (B2') on the reporter protein interacts with the respective binding moiety (B2") on the second regulator moiety, thereby co-localizing the reporter protein with the first and second regulator moieties and facilitating binding of the first regulator moiety to the first heterologous amino acid sequence comprised in the reporter protein and binding of the second regulator moiety to the second heterologous amino acid sequence comprised in the reporter protein. The binding of the first regulator moiety to the first heterologous amino acid sequence and the second regulator moiety to the second heterologous amino acid sequence (typically simultaneously, concomitantly) typically induces a conformational change in the reporter protein, thereby reversibly modulating the activity of the reporter protein. Thus, finally, the presence of the first target molecule TM1 and the second target molecule TM2 reversibly modulates the activity of the reporter protein.

[0081] The activity of the reporter protein can then be detected by appropriate methods, depending on the identity of the reporter protein, to provide a readout of the presence of one or more target molecules.

[0082] Compositions, kits and detection devices As described above, provided herein is a biosensor comprising (i) a reporter protein as described herein; (ii) a first regulator portion as described herein; and (iii) a second regulator portion as described herein. Also described herein is a composition or kit comprising (a) a reporter protein as described herein; or (b) a biosensor as described herein. When the reporter protein is an enzyme, the composition or kit may further comprise a substrate for the enzyme, as further described herein.

[0083] The composition or kit may further comprise one or more target molecules. For example, the composition or kit may further comprise a first target molecule (TM1) and / or a second target molecule (TM2). Suitable target molecules are described further herein, but may preferably be selected from methotrexate, phenol glucoside, rapamycin, tacrolimus, cyclosporine A. The composition or kit may further comprise a first kind of target molecule, for example, for use as a positive control (e.g., TM1=TM2, which are the same). The composition or kit may further comprise two kinds of target molecules, for example, for use as a positive control (i.e., TM1 and TM2, which are different).

[0084] The composition or kit may further comprise a sample, typically a sample provided by a user and added to the provided composition or kit. The sample may be suspected to contain one or more target molecules or may be tested for the presence of one or more target molecules. Thus, the sample may or may not contain the target molecule TM1 and / or the target molecule TM2, or it may be unknown whether the sample contains TM1 and / or TM2. The sample may be a biological sample or an environmental sample, preferably a biological sample. A biological sample may be understood to mean a sample derived from an organism, preferably a sample obtained from a living organism. The biological sample may be obtained from an animal (preferably a mammal, most preferably a human), a plant or a fungus. The sample may be obtained from an animal or a plant of agricultural importance, such as livestock (e.g., cows, sheep, pigs, chickens, etc.) or crops (e.g., vegetables, grains, or other food products). The sample may be a biological sample, the biological sample being selected from tissue, blood, plasma, serum, saliva, interstitial fluid, milk, juice, sap or homogenate. An environmental sample may be understood to mean a sample of inorganic origin, such as a water sample, a rock sample, or an air sample. A person skilled in the art will be readily able to prepare a particular sample for analysis using the compositions and kits and / or following the methods described herein.

[0085] A composition or kit comprising a reporter protein or biosensor of the present invention may further comprise a second enzyme, also referred to herein as an adaptor enzyme. The terms "second enzyme" and "adaptor enzyme" may be used interchangeably. When the reporter protein is an enzyme, the composition or kit may further comprise an adaptor enzyme (i.e., a second enzyme). The role of the adaptor enzyme is typically to convert the product of the biosensor enzyme into a readout that is easier or simpler to detect. Thus, typically, the product of the biosensor enzyme is a substrate for the adaptor enzyme. For example, the biosensor enzyme may be trehalase, which catalyzes the conversion of trehalose to glucose. The adaptor enzyme may then be, for example, a GDH enzyme, which converts glucose to gluconolactone and electrons. The electrons produced by the GDH enzyme can then be easily measured.

[0086] In some examples, the adapter enzyme may comprise a heterologous amino acid sequence that is responsive to the peptide, and binding of the peptide to the heterologous amino acid sequence reversibly modulates the catalytic activity of a second enzyme, and the substrate of the adapter enzyme is the catalytic product of the reporter protein as described herein, where the reporter protein is an enzyme. The adapter enzyme is typically a fast enzyme, preferably a diffusion-limited enzyme. The adapter enzyme may be an oxidoreductase as described herein, preferably GDH, most preferably PQQ-GDH. The adapter enzyme may be a β-lactamase as described herein. The adapter enzyme may be a carbonic anhydrase as described herein, and the readout of the enzyme activity, and thus the biosensor, may be a change in pH. The heterologous amino acid sequence is typically provided as an insertion within the amino acid sequence of the adapter enzyme. The heterologous amino acid sequence may be a calmodulin protein or a functional fragment thereof as described herein. The peptide may be a calmodulin-binding peptide (CaM-BP) as described herein. Thus, the composition or kit may further comprise a GDH enzyme comprising a calmodulin protein or a functional fragment thereof responsive to a calmodulin-binding peptide (CaM-BP), the binding of CaM-BP to the calmodulin protein or a functional fragment thereof reversibly activates the catalytic activity of the GDH enzyme, and optionally the catalytic product of the reporter protein described herein is a substrate for the GDH enzyme. The calmodulin protein or a functional fragment thereof may be inserted into the sequence of the GDH enzyme, preferably into the loop connecting β-sheets 5 and 6, most preferably at a position corresponding to positions 403-405 (amino acid residues Ser403-Asn405) of PQQ-GDH of SEQ ID NO: 5. The adaptor protein comprising a heterologous amino acid sequence may comprise or essentially consist of the sequence of SEQ ID NO: 63.

[0087] In some examples, the adapter enzyme comprising a heterologous amino acid sequence responsive to the peptide may further comprise a binding moiety (AB') and the peptide may further comprise a respective binding moiety (AB"). The binding moieties (AB' and / or AB") may be as described herein. The two binding moieties typically form a binding moiety pair, as described herein. The binding moiety pair (AB' and AB") typically binds to a target molecule, preferably a target molecule (TM AB ) and thus interact with the target molecule (TM AB The presence of the binding moieties (AB' and AB'') allows the peptide to interact with the adaptor enzyme, thereby colocalizing the peptide with the adaptor enzyme, which then binds to the heterologous amino acid sequence and activates the catalytic activity of the adaptor enzyme. Thus, activation of the catalytic activity of the adaptor enzyme is achieved by binding the target molecule (TM AB ), which may be as described herein. Preferably, the target molecule of the adaptor enzyme (TM AB ) are different from the target molecules (TM1 and TM2) of the reporter proteins described herein comprised in the biosensors of the invention or in the compositions or kits of the invention. Thus, the binding moiety pair (AB' and AB'') associated with the adaptor enzyme is typically different from the binding moiety pair (B1' and B1'', and B2' and B2'') associated with the reporter enzymes described herein. Thus, the inclusion of an adaptor enzyme may add another input to the biosensor system. For example, a signal from an adaptor enzyme may be detected in the presence of (i) one or more target molecules (TM1 and TM2) of the reporter proteins / biosensors of the invention described herein, and (ii) a target molecule (TM AB When the reporter protein is an enzyme, activation of the reporter protein's activity is necessary to produce a reporter protein product, which is typically a substrate for the adaptor enzyme.

[0088] Also described herein is a detection device comprising a cell or chamber comprising a reporter protein, biosensor, or composition described herein. Suitably, a sample can be introduced into the cell or chamber to facilitate detection of one or more target molecules (e.g., TM1 and TM2). The sample can be, for example, a biological sample or an environmental sample, as described herein. In certain examples, the detection device can provide an electrochemical, acoustic, and / or optical signal indicative of the presence of one or more target molecules (e.g., TM1 and TM2). In some examples, the detection device can comprise an electrode. In some examples, the detection device can comprise a semiconductor device. In some examples, the detection device is a device adapted for amperometry. The device can comprise a screen-printed electrode, preferably laminated with a dry mixture comprising a biosensor of the present invention and preferably an electron mediator.

[0089] The detection device may further provide a disease diagnosis from the diagnostic subject results by comprising a processor and a memory coupled to the processor, the memory containing computer readable program code components that, when executed by the processor, perform a set of functions including analyzing the diagnostic test results and providing a diagnosis of a disease or condition.

[0090] The detection device may further provide for communicating diagnostic test results by comprising a processor and a memory coupled to the processor, the memory containing computer readable program code components that, when executed by the processor, perform a set of functions including: transmitting the diagnostic result to a receiving device; and, optionally, receiving a diagnosis of the disease or condition from the receiving device or another receiving device.

[0091] Preferred biosensors As will be understood, any of the above components of the reporter protein / biosensor may be combined together to form the reporter protein / biosensor of the present invention. In this section, we describe some of the most preferred reporter protein / biosensor architectures, which are provided to further illustrate, but are not limited to, the function of the reporter protein / biosensor of the present invention and the reporter protein / biosensor described herein.

[0092] Thus, also described herein is a reporter protein which is an enzyme or a fluorescent protein, comprising a first calmodulin protein sequence responsive to binding of a first CaM-BP and a second calmodulin protein sequence responsive to binding of a second CaM-BP, wherein binding of the first CaM-BP to the first calmodulin protein sequence and binding of the second CaM-BP to the second calmodulin protein sequence reversibly modulates the activity of the reporter protein, and wherein the first calmodulin protein sequence and the second calmodulin protein sequence are provided as insertions within the amino acid sequence of the reporter protein, preferably inserted at different positions within the amino acid sequence of the reporter protein. Preferably, the reporter protein is an enzyme.

[0093] Also described herein is a reporter protein which is an enzyme or a fluorescent protein, comprising a first calmodulin protein sequence responsive to binding of a first CaM-BP and a second calmodulin protein sequence responsive to binding of a second CaM-BP, wherein binding of the first CaM-BP to the first calmodulin protein sequence and binding of the second CaM-BP to the second calmodulin protein sequence reversibly modulates the activity of the reporter protein, the first and second calmodulin protein sequences being provided as insertions within the amino acid sequence of the reporter protein, and the reporter protein further comprising two binding moieties (B1' and B2'). Preferably, the binding moieties B1' and B2' are both proteins. The binding moieties B1' and B2'' may bind to the same species of target molecule (TM1 = TM2). The amino acid sequences of the binding moieties B1' and B2' may be the same. The first and second CaM-BPs may each further comprise a respective binding moiety (B1" and B2", B1"=B2") that forms a binding pair with a binding moiety on the reporter protein such that the binding moiety (B1" and B2") on the CaM-BP can interact with the binding moiety (B1' and B2') on the reporter protein via a single species of target molecule (TM1=TM2). Alternatively, the binding moieties B1' and B2" may bind to different species of target molecules (TM1 and TM2, respectively). The amino acid sequences of the binding moieties B1' and B2' may be different. The first CaM-BP may further comprise a binding moiety (B1'') that forms a binding pair with a binding moiety (B1') on the reporter protein, and the second CaM-BP may further comprise a respective binding moiety (B2'') that forms a binding pair with a binding moiety (B2') on the reporter protein, such that the two binding moiety pairs interact via two different species of target molecules, with B1' and B1'' interacting via TM1 and B2' and B2'' interacting via TM2.

[0094] Also described herein is a reporter protein which is an enzyme or a fluorescent protein, comprising a first calmodulin protein sequence responsive to binding of a first CaM-BP and a second calmodulin protein sequence responsive to binding of a second CaM-BP, wherein binding of the first CaM-BP to the first calmodulin protein sequence and binding of the second CaM-BP to the second calmodulin protein sequence reversibly modulates the activity of the reporter protein, the first and second calmodulin protein sequences being provided as insertions within the amino acid sequence of the reporter protein, and the reporter protein further comprising a binding moiety (B1'). Preferably, the binding moiety B1' is a protein. The first and second CaM-BPs are linked (preferably, the first and second CaM-BPs are comprised within the same contiguous amino acid sequence) and further comprise respective binding moieties (B1'') that form a binding pair with a binding moiety (B1') on the reporter protein (preferably, further comprised within the same contiguous amino acid sequence), such that the binding moiety (B1'') on the CaM-BP may be capable of interacting with the binding moiety (B1') on the reporter protein via a single species of target molecule (TM1). Also described herein is a biosensor comprising a first component and a second component, the first component being a reporter protein comprising a first calmodulin protein sequence responsive to binding of a first CaM-BP and a second calmodulin protein sequence responsive to binding of a second CaM-BP, wherein binding of the first CaM-BP to the first calmodulin protein sequence and binding of the second CaM-BP to the second calmodulin protein sequence reversibly modulates activity of the reporter protein, the first and second calmodulin protein sequences being provided as insertions within the amino acid sequence of the reporter protein, the reporter protein further comprising a binding moiety (B1'); the second component comprising a binding moiety (B1'') capable of interacting with the binding moiety (B1') on the reporter protein linked to the first CaM-BP and the second CaM-BP. Optionally, the first and second CaM-BPs have reduced affinity for calmodulin compared to a wild-type CaM-BP.Alternatively, the CaM-BP is a caged CaM-BP, i.e., each is further linked to a calmodulin protein that has a reduced binding affinity for the CaM-BP compared to the binding affinity of the calmodulin insert on the reporter protein for the CaM-BP.

[0095] Thus, in a preferred example, (i) the heterologous amino acid sequences (e.g., the first and second amino acid sequences, and any further heterologous amino acid sequences) are each a calmodulin protein or a variant or functional fragment thereof, (ii) the regulator moieties (e.g., the first and second regulator moieties) are peptides, preferably CaM-BP, (iii) the reporter protein is an enzyme or a fluorescent protein, (iv) the reporter protein comprises two heterologous amino acid sequences, (v) the reporter protein comprises two binding domains (e.g., the first and second binding moieties B1' and B2'), and (vi) the reporter protein / biosensor targets one or two different target molecules, depending on whether the two binding moiety pairs are the same or different.

[0096] In some examples, a reporter protein comprising a first heterologous amino acid sequence and a second heterologous amino acid sequence comprises or consists of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to any one of SEQ ID NOs: 51, 52, 57, 62, 75, 81, 86 or 89. In some examples, a reporter protein comprising a first heterologous amino acid sequence and a second heterologous amino acid sequence comprises or consists of any one of SEQ ID NOs: 51, 52, 57, 62, 75, 81, 86 or 89, or a variant thereof. In some examples, the reporter protein comprising the first heterologous amino acid sequence and the second heterologous amino acid sequence comprises or consists of any one of SEQ ID NOs: 51, 52, 57, 62, 75, 86, or 89, and one or both of the calmodulin insertions are replaced with a different heterologous amino acid sequence, such as an affinity clamp or SBP, as described herein. In some examples, the reporter protein comprising the first heterologous amino acid sequence and the second heterologous amino acid sequence comprises or consists of SEQ ID NO: 81, and one or both of the affinity clamp insertions are replaced with a different heterologous amino acid sequence, such as a calmodulin protein or SBP, as described herein.

[0097] A preferred biosensor typically comprises the preferred reporter protein described above. The biosensor may be configured for the detection of rapamycin, and may comprise (i) a reporter protein (which may be an enzyme or a fluorescent protein, and may optionally be a trehalase enzyme, a GDH enzyme, or a β-lactamase enzyme) that comprises two calmodulin protein insertions at different positions in the reporter protein amino acid sequence and two FKBP binding moieties, one at the N-terminus and one at the C-terminus of the reporter protein amino acid sequence; and (ii) a regulatory moiety that comprises a calmodulin binding peptide and an FRB binding moiety. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme. When the reporter protein is trehalase, the biosensor may further comprise the enzyme substrate trehalose. When the reporter protein is β-lactamase, the biosensor may further comprise the enzyme substrate nitrocefin. Thus, a biosensor for detecting rapamycin may comprise a first component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably 80%, most preferably 90% sequence identity to SEQ ID NO: 53 or 58; and a second component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO: 65.

[0098] The biosensor may be configured for the detection of tacrolimus, and may comprise (i) a reporter protein (which may be an enzyme or a fluorescent protein, and optionally a trehalase enzyme, a GDH enzyme, or a β-lactamase enzyme) comprising two calmodulin protein insertions at different positions within the reporter protein amino acid sequence and two FKBP binding moieties, one at the N-terminus and one at the C-terminus of the reporter protein amino acid sequence; and (ii) a regulatory moiety comprising a calmodulin binding peptide and a calcineurin A and B (CalA / B) binding moiety. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme. Thus, a biosensor for detecting tacrolimus may comprise a first component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably 80%, most preferably 90%, sequence identity to SEQ ID NO: 53 or 58; and a second component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO: 66.

[0099] The biosensor may be configured for the detection of methotrexate (MTX), and may comprise (i) a reporter protein (which may be an enzyme or a fluorescent protein, and optionally a trehalase enzyme, a GDH enzyme, or a β-lactamase enzyme) comprising two calmodulin protein insertions at different positions within the reporter protein amino acid sequence and two MTX-specific VHH binding moieties, one at the N-terminus and one at the C-terminus of the reporter protein amino acid sequence; and (ii) a regulatory moiety comprising a calmodulin-binding peptide and an MTX-specific nanoCLAMP binding moiety. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme. Thus, a biosensor for detecting MTX may comprise a first component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably 80%, most preferably 90%, sequence identity to any one of SEQ ID NOs: 59, 84 or 87; and a second component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, most preferably at least 90%, sequence identity to SEQ ID NOs: 67 or 88.

[0100] The biosensor may be configured for the detection of rapamycin and cyclosporine, and may comprise: (i) a reporter protein (which may be an enzyme or a fluorescent protein, and may optionally be a trehalase enzyme, a GDH enzyme, or a β-lactamase enzyme) comprising two calmodulin protein insertions at different positions within the reporter protein amino acid sequence; optionally an FKBP binding moiety at the N-terminus of the reporter protein amino acid sequence; and optionally a cyclophilin binding moiety at the C-terminus of the reporter protein amino acid sequence; (ii) a regulatory moiety comprising a calmodulin binding peptide and an FRB binding moiety; and (iii) a regulatory moiety comprising a calmodulin binding peptide and a calcineurin A and B (CalA / B) binding moiety. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme. When the reporter protein is trehalase, the biosensor may further comprise the enzyme substrate trehalose. Thus, a biosensor for detecting rapamycin may comprise a first component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably 80%, most preferably 90% sequence identity to SEQ ID NO:54; a second component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO:65; and a third component comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, preferably at least 80%, most preferably at least 90% sequence identity to SEQ ID NO:66.

[0101] The biosensor may be configured for the detection of a protease, the biosensor may comprise: (i) a reporter protein (which may be an enzyme or a fluorescent protein, and optionally a trehalase enzyme, a GDH enzyme or a β-lactamase enzyme) comprising two calmodulin protein insertions at different positions within the reporter protein amino acid sequence; and (ii) a regulatory moiety comprising a CaM-BP linked via a peptide linker comprising a cleavage site of a target protease to a calmodulin protein having reduced binding affinity for the CaM-BP compared to the binding affinity of the calmodulin protein insertions. When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme.

[0102] The biosensor may be configured to detect a target molecule (TM1), the biosensor comprising: (1) a reporter protein (which may be an enzyme or a fluorescent protein, and optionally a trehalase enzyme, a GDH enzyme, or a β-lactamase enzyme) comprising two calmodulin protein insertions at different positions within the reporter protein amino acid sequence and a first binding moiety (B') specific for TM1, optionally at the N-terminus or C-terminus of the reporter protein amino acid sequence; and (2-i) two CaM-BPs (optionally each CaM-BP is calmodulin-specific relative to a wild-type CaM-BP). or (2-ii) a regulatory moiety comprising a second binding moiety (B') that is also specific for TM1 or specific for the complex of TM1 and B' linked to a caged CaM-BP (each caged CaM-BP comprising a CaM-BP linked to a calmodulin protein that has reduced binding affinity for the CaM-BP compared to the binding affinity of the calmodulin protein insert of the reporter protein), or (2-iii) a regulatory moiety comprising a second binding moiety (B') that is also specific for TM1 or specific for the complex of TM1 and B' linked to two caged CaM-BPs (each caged CaM-BP comprises a CaM-BP linked to a calmodulin protein that has reduced binding affinity for the CaM-BP compared to the binding affinity of the calmodulin protein insert of the reporter protein). When the reporter protein is an enzyme, the biosensor may further comprise a substrate for the enzyme.

[0103] cascade Two or more biosensors as described herein can be used in tandem to generate a cascade, e.g., an enzymatic cascade, in which multiple steps can be regulated. Two or more biosensors can be used at directly successive steps in the cascade or at indirectly successive steps (e.g., one or more intervening steps are not monitored by a biosensor).

[0104] For example, trehalose can be converted to glucose using a first biosensor as described herein, and glucose can be converted to gluconolactate using a second biosensor. The first biosensor can be any biosensor that includes trehalase. Such a first biosensor can be configured to respond to rapamycin, for example, the first biosensor can include a reporting protein that includes or consists of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO:54, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to SEQ ID NO:65, and optionally at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO:54, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to SEQ ID NO:65. It also includes a first heterologous amino acid sequence comprising or consisting of a sequence having 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity, and a second heterologous amino acid sequence comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity to SEQ ID NO:66.

[0105] The second biosensor can be any biosensor that contains GDH. Such a second biosensor can be configured to respond to cyclosporine A, for example, the second biosensor can include a reporting protein that comprises or consists of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO: 64, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity, and optionally also includes a first heterologous amino acid sequence and a second heterologous amino acid sequence that comprises or consists of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO: 66, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity. Alternatively, such a second biosensor may be configured to respond to methotrexate (MTX), for example, the second biosensor may comprise a reporting protein comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO: 84, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity, and optionally also comprising the first heterologous amino acid sequence and a second heterologous amino acid sequence comprising or consisting of a sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or at least 100% sequence identity to SEQ ID NO: 67 or 88, optionally at least 80%, preferably at least 90%, most preferably at least 95% sequence identity.

[0106] One of skill in the art will understand that any of the biosensors described herein can be configured to form a cascade.

[0107] method Also described herein is a method for detecting one or more target molecules (preferably one target molecule or two target molecules) (e.g., TM1 and TM2), comprising contacting one or more of the biosensors described herein with a sample under conditions suitable for detecting the presence or absence of one or more target molecules in the sample. Also described herein is a method for detecting one or more target molecules (preferably one target molecule or two target molecules) (e.g., TM1 and TM2), comprising contacting one or more of the reporter proteins described herein with a sample under conditions suitable for detecting the presence or absence of one or more target molecules in the sample.

[0108] The sample may be any sample suspected of containing one or more target molecules. The sample may or may not contain one or more target molecules (e.g., target molecule TM1 and / or target molecule TM2 as described herein). The sample may be a biological sample or an environmental sample. The sample is preferably a biological sample. A biological sample may be understood to mean a sample derived from an organism, preferably a sample obtained from a living organism. A biological sample may include an organ sample, a tissue sample, a cell sample, a fluid sample, or any other sample obtainable, obtained, derivable, or derived from an organism or a component of an organism. For example, a biological sample may include a fermentation medium, a feedstock, or a food product, such as, but not limited to, a dairy product. Suitably, the enzymatic activity of the biosensor is not substantially inhibited by the components of the sample (e.g., serum proteins, metabolites, cells, cell debris and components, naturally occurring protease inhibitors, etc.). The biological sample may be obtained from an animal (preferably a mammal, most preferably a human), a plant, or a fungus. The sample may be obtained from an agriculturally important animal or plant, such as livestock (e.g., cows, sheep, pigs, chickens, etc.) or crops (e.g., vegetables, grains, or other food products). Preferably, the sample may be a biological sample, the biological sample being selected from tissue, blood, plasma, serum, saliva, interstitial fluid, milk, juice, sap, or homogenate. The biological sample may be obtainable or obtained from a mammal, preferably a human. The biological sample may be a fluid sample, such as blood, serum, plasma, urine, saliva, tears, sweat, cerebrospinal fluid, or amniotic fluid. The sample may be a tissue sample, such as a tissue or organ biopsy, or a cell sample, such as a sample containing red blood cells, lymphocytes, tumor cells, or skin cells. A particular type of biological sample is a pathology sample. The sample may be a blood, saliva, serum, or urine sample from a human subject. The sample may be a blood or saliva sample obtained from a human. The sample may be a serum or urine sample from a human subject. The human may be a patient.A human may have or be suspected of having a disease for which one or more target molecules are markers or biomarkers.An environmental sample may be understood to mean a sample of inorganic origin, such as a water sample, a soil sample, a rock sample or an air sample.

[0109] In some examples, the biosensor and / or method of use may be applicable to drug testing, for example to detect the use of illegal drugs of addiction (e.g., cannabinoids, amphetamines, cocaine, heroin, etc.) and / or for the detection of performance enhancing substances in sports and / or masking agents commonly used to avoid detection of performance enhancing substances, which may be applicable to the detection of banned performance enhancing substances in humans and / or other mammals, such as race horses and greyhounds, that may be subject to illegal "doping" to enhance performance.

[0110] The biosensor of the present invention can also be used to screen proteins that bind to a specific target molecule. For example, in some instances, the sample can contain a known purified target molecule, such as a target protein, a target peptide, or a target small molecule. A plurality of biosensors can be provided that contain a panel of different binding moiety pairs that are screened for binding to one or more target molecules. As described herein, the specific binding of the binding moiety pair to the target molecule results in the activation of the biosensor.

[0111] Also described herein is a method for diagnosing a disease or condition in an organism, the method comprising contacting one or more reporter proteins as described herein, or one or more biosensors as described herein, with a sample obtained from the organism under conditions suitable for detecting the presence or absence of one or more target molecules in the sample, the presence or absence of one or more target molecules in the sample indicating whether the organism has or is at risk of having a disease or condition. Typically, the one or more target molecules are either one target molecule or two target molecules. In some examples, determining the presence or absence of one or more target molecules facilitates the diagnosis of a disease or condition. The organism may include plants and animals, including mammals such as fish, birds, and humans. Preferably, the organism is a mammal, most preferably a human.

[0112] The disease or condition may be any disease or condition for which detection of the target molecule aids in diagnosis. Suitable target molecules are described herein. The target molecules may include one or more of the previously described kallikreins, including PSA, matrix metalloproteinases, viral and bacterial proteases, antibodies, glucose, triglycerides, lipoproteins, cholesterol, tumor antigens, lymphocyte antigens, autoantigens and autoantibodies, blood clotting factors such as drugs, salts, creatinine, serum or plasma proteins, pesticides, uric acid, products and intermediates of human and animal metabolism, and metals. The target molecules preferably include one or more of methotrexate, phenol glucoside, rapamycin, tacrolimus, or cyclosporin A. These methods may be adapted to be performed as "point-of-care" methods, whereby the determination of the presence or absence of the target molecule is made at the patient's location and then analyzed at that location or transmitted to a remote location for diagnosis of the disease or condition.

[0113] Also described herein is a method of monitoring one or more target molecules in an organism, comprising contacting one or more reporter proteins as described herein, or one or more biosensors as described herein, with a sample obtained from the organism under conditions suitable for detecting the presence or absence of one or more target molecules in the sample. Multiple samples may be taken at regular intervals over a defined period, such as over a day, a week, a month, or a year, to monitor the presence or absence of the target molecules in the organism over the period. The method may further comprise quantifying the level of the target molecules in the organism. The method may further comprise detecting and quantifying the target molecules in the organism. The method may further comprise monitoring the level of one or more target molecules in the organism over a period, for example, which may be a day, a week, a month, a year, or multiple years. The method may allow different states of normal physiology to be monitored in the organism. Thus, the presence or absence of one or more target molecules in the sample may indicate a particular physiological state in the organism. The level (ie, amount or concentration) of one or more target molecules in a sample can be indicative of a particular physiological state in an organism.

[0114] In some embodiments, the method may be for monitoring a fertility cycle, and the one or more target molecules are hormones, such as progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH). The method may be for monitoring a level of psychological stress, and the one or more target molecules are cortisol or alpha-amylase. The method may be for monitoring a metabolic state, and the one or more target molecules are insulin and glucose. The method may be for monitoring a level of a therapeutic drug, such as methotrexate, rapamycin, tacrolimus, or cyclosporine A. The method may be for monitoring a level of an environmental contaminant, such as a phenolic glucoside. The organism may be a plant (such as an agriculturally important plant, such as a crop) or an animal, such as a fish, a bird, or a mammal (such as an agriculturally important animal, such as livestock). The organism may be a mammal. Preferably, the organism is a human.

[0115] Also described herein is a kit or device adapted to carry out the methods described herein. Described herein is a kit that includes one or more different biosensors as described herein that can detect one or more different target molecules. In this regard, the kit can include an array of different biosensors as described herein that can detect multiple different target molecules. The kit can further include one or more suitable substrates of the reporter protein(s) of the biosensor as described herein. The kit can also include additional components, including reagents such as buffers and diluents, reaction vessels, and instructions for use.

[0116] In some examples, the reporter proteins or biosensors described herein can be used to assay for protein-protein or protein-small molecule binding interactions. Thus, described herein is a method for assaying protein-protein or protein-small molecule interactions, comprising contacting a reporter protein or biosensor described herein with a sample under conditions suitable for detecting the presence or absence of an interaction between a pair of binding moieties or between a pair of binding moieties and their corresponding target molecules. The sample typically includes a substrate molecule suitable for the reporter protein or biosensor.

[0117] In the biosensors of the present invention, the activity of the reporter protein may depend on a specific protein-protein or protein-small molecule interaction between the binding moiety pair (i.e., no target molecule is required). Such biosensors may be used to assay for direct interactions between a first protein or small molecule of interest (i.e., one half of the binding moiety pair) and a second protein or small molecule of interest (i.e., the corresponding other half of the binding moiety pair). The direct interaction between the protein / small molecule of interest at the binding moiety on the reporter protein and the binding corresponding binding moiety on the regulatory moiety (forming a binding moiety pair) colocalizes the reporter protein with the regulatory moiety and promotes binding of the regulatory moiety to the heterologous amino acid sequence of the reporter protein, thereby activating the activity of the reporter protein described herein to produce a detectable readout as described herein. Such biosensors may be used to assay for direct interactions between the two halves of each binding moiety pair in the biosensor. Such biosensors may further be used to assay for activators and inhibitors of the interaction between the two halves of the binding moiety pair, which may be proteins or small molecules. Thus, the sample may further contain putative activators and inhibitors, i.e., molecules that are assayed for their ability to activate or inhibit the interaction between the binding moiety pair. Activators that enhance the interaction between the binding moiety pair will result in an increase in the activity of the reporter protein. Inhibitors that inhibit, prevent or reduce the interaction between the binding moiety pair will result in a decrease in the activity of the reporter protein.

[0118] Also described herein is a method for converting an enzyme into an enzyme whose catalytic activity is reversibly controlled depending on the presence of one or more target molecules. The method may comprise, in a first step (a), generating a library of single-insertion enzyme mutants by inserting heterologous amino acid sequences responsive to the binding of a regulator moiety at several different positions in the enzyme sequence.

[0119] Single insertion enzyme variants can be generated by introducing a heterologous amino acid sequence at a specific position within the amino acid sequence of the enzyme. Typically, the heterologous amino acid sequence is inserted at a specific position within the amino acid sequence of the enzyme, typically between two consecutive residues within the amino acid sequence of the enzyme. A person skilled in the art would be readily able to perform such insertions using, for example, recombinant DNA manipulations (e.g., using restriction enzymes, ligation, and PCR techniques) and recombinant protein expression techniques well known in the art. A library of single insertion enzyme variants can be generated, with each single enzyme variant comprising one heterologous amino acid sequence inserted at a specific position. Typically, each single insertion enzyme variant in the library comprises a heterologous amino acid sequence inserted at a different position within the amino acid sequence of the enzyme. The enzyme and heterologous amino acid sequences are typically provided as contiguous amino acid sequences. For example, a single insertion enzyme variant may comprise, from N-terminus to C-terminus, a first portion of the enzyme sequence, a heterologous amino acid sequence, and a second portion of the enzyme sequence. Typically, the first and second portions of the enzyme sequence substantially correspond to the amino acid sequence of the wild-type enzyme. The heterologous amino acid sequence that is responsive to binding of the modulator moiety can be any heterologous amino acid sequence described herein. Preferably, the heterologous amino acid sequence is a calmodulin protein or a functional fragment thereof as described herein, and the modulator moiety is a calmodulin-binding peptide (CaM-BP) as described herein.

[0120] The method may further comprise assaying the catalytic activity of the library of single insertion enzyme variants. Thus, the catalytic activity of each single insertion enzyme variant in the library may be assayed. Such an assay may comprise contacting the single insertion enzyme variant with its substrate under conditions suitable for the catalytic activity of the enzyme. The signal or readout of the catalytic activity of the enzyme may be detected, measured and / or quantified. A person skilled in the art will be able to readily select an appropriate assay for detecting, measuring and / or quantifying the catalytic activity of the enzyme. Typically, the activity assay is performed in the presence and absence of a regulator moiety. The method may further comprise selecting a single insertion enzyme variant that exhibits a change in catalytic activity upon binding of the regulator moiety, usually in the presence of a suitable enzyme substrate and under suitable reaction conditions. Preferably, a single insertion enzyme variant that exhibits a change in catalytic activity upon binding of the regulator moiety within a desired time frame. For example, a single insertion enzyme variant that exhibits a fast response time to the addition of the regulator moiety is desirable. The selected single insertion enzyme variant may exhibit a change in catalytic activity (i.e., a significant change, such as at least twice the catalytic activity in the absence of the regulator moiety) within 30 seconds of the addition of the regulator moiety. The single insertion enzyme variants preferably have a dynamic range of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or at least 20 fold, preferably 2-20 fold, most preferably 5-15 fold. The desired time frame can be less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than 1 minute, most preferably less than 10 minutes. The change in catalytic activity that occurs within the desired time frame is typically at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold the catalytic activity (i.e., signal change) in the absence of the regulator moiety, preferably the change in catalytic activity that occurs within the desired time frame is at least 5-fold, most preferably at least 10-fold.

[0121] The method may further comprise step (b) of generating a library of double insertion enzyme variants by inserting an additional heterologous amino acid sequence that is responsive to binding of the regulator moiety at a second site within the enzyme sequence. A double insertion enzyme variant may typically be understood to comprise two heterologous amino acid sequences inserted into the amino acid sequence of the enzyme, typically at different positions within the enzyme amino acid sequence. The enzyme and the two heterologous amino acid sequences are typically provided as contiguous amino acid sequences. For example, a double insertion enzyme variant may comprise, from N-terminus to C-terminus, a first portion of the enzyme sequence, a first heterologous amino acid sequence, a second portion of the enzyme sequence, a second heterologous amino acid sequence, and a third portion of the enzyme sequence. Typically, the first, second and third portions of the enzyme sequence substantially correspond to the amino acid sequence of the wild-type enzyme. Each heterologous amino acid sequence may be a heterologous amino acid sequence as described herein. The two heterologous amino acid sequences may be the same or different. Each of the two heterologous amino acid sequences may comprise or consist of a calmodulin protein or a functional fragment thereof. A library of double insertion enzyme variants typically includes a plurality of double insertion enzyme variants, each of which contains two heterologous amino acid sequences inserted at different positions. For each double insertion enzyme variant, the insertion position of one of the heterologous amino acid sequences may be the same, and the insertion position of the other may be different. Or, for each double insertion enzyme variant, the insertion positions of the two heterologous amino acid sequences may both be different. In some instances, the insertion positions of the double insertion mutants may be selected by combining the insertion positions found to have the fastest response time of the single insertion enzyme variants.

[0122] The method may further comprise the step of selecting a double insertion enzyme mutant that exhibits a change in catalytic activity in the presence of a regulator moiety and an increased dynamic range within a desired time frame compared to the corresponding single insertion enzyme mutant. Thus, a double insertion enzyme mutant is selected that exhibits a change in catalytic activity upon addition of a regulator moiety, usually in the presence of an enzyme substrate and under suitable reaction conditions. As mentioned above, a person skilled in the art will be able to easily select an appropriate assay for the catalytic activity of a particular enzyme. The change in catalytic activity upon addition of a regulator moiety of a double insertion mutant enzyme may be compared to the change in catalytic activity upon addition of a regulator moiety of a corresponding single insertion mutant enzyme, i.e., a single insertion mutant enzyme having a heterologous amino acid sequence inserted at a position corresponding to one of the two positions where the heterologous amino acid sequence is inserted in the double insertion mutant enzyme. Preferably, a double insertion mutant enzyme is selected that exhibits an improved dynamic range within a desired time frame compared to either, preferably both, of the single insertion mutant enzymes. The dynamic range of the double insertion mutant enzyme may be at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 90-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold or 100-fold, preferably at least 10-fold, most preferably at least 25-fold, of the dynamic range of either, preferably both, of the single insertion mutant enzymes. The desired time frame may be less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes, or less than 1 minute, most preferably less than 10 minutes. The change in catalytic activity that occurs within the desired time frame is typically at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold the catalytic activity (i.e., signal change) in the absence of the modulator moiety, preferably the change in catalytic activity that occurs within the desired time frame is at least 5-fold, most preferably at least 10-fold. Thus, preferably, for example for clinical applications, a reporter protein or biosensor described herein can exhibit at least a 5-fold signal change (i.e., change in catalytic activity upon addition of a modulator moiety compared to catalytic activity in the absence of the modulator moiety) within 20 minutes, and most preferably, a reporter protein or biosensor described herein can exhibit at least a 10-fold signal change within 10 minutes.

[0123] Thus, there is provided a method for converting a constitutively active enzyme into a reversibly regulated enzyme whose catalytic activity is dependent on the presence of one or more target molecules, comprising: (a) generating a library of single insertion enzyme mutants by inserting heterologous amino acid sequences responsive to binding of a regulatory factor moiety at several different positions within the enzyme sequence, and selecting single insertion enzyme mutants that exhibit a change in catalytic activity within a desired time frame upon binding of the regulatory factor moiety; (b) generating a library of double insertion enzyme mutants by inserting an additional heterologous amino acid sequence at a second site within the enzyme sequence that is responsive to binding of the regulator moiety, and selecting double insertion enzyme mutants that exhibit a change in catalytic activity in the presence of the regulator moiety and have an increased dynamic range compared to the corresponding single insertion enzyme mutants (preferably the double mutant insertion enzymes also exhibit a change in catalytic activity within a desired time frame). A method is described herein, comprising:

[0124] nucleic acid The present invention also provides one or more isolated nucleic acids encoding the reporter proteins of the present invention, the biosensors of the present invention, or components of the biosensors of the present invention. The nucleic acids may encode any of SEQ ID NOs: 1-67, 69, 70, or 73-82, or variants thereof. The nucleic acids may encode an amino acid sequence comprising one or more of SEQ ID NOs: 1-67, 69, 70, or 73-82, or any of the sequences. The nucleic acids may comprise SEQ ID NO: 68 or SEQ ID NO: 71, or variants thereof.

[0125] Described herein are one or more genetic constructs comprising one or more isolated nucleic acids of the invention. Described herein are one or more vectors comprising one or more isolated nucleic acids of the invention. Described herein are one or more vectors suitable or adapted for expression of, for example, a reporter protein described herein, a biosensor described herein, or a component of a biosensor described herein, in a suitable host cell (see, for example, SEQ ID NO: 68 or SEQ ID NO: 71). Described herein are host cells comprising one or more isolated nucleic acids, one or more genetic constructs, or one or more vectors described herein (see, for example, Figures 15-18).

[0126] The term "nucleic acid" as used herein refers to single-stranded or double-stranded mRNA, RNA, cRNA, RNAi, siRNA and DNA, including cDNA, mitochondrial DNA (mtDNA) and genomic DNA. Preferably, the nucleic acid of the present invention is DNA. The present invention also provides isolated variants and / or fragments of nucleic acids. Variants may include nucleotide sequences with at least 70%, at least 75%, preferably at least 80%, at least 85%, more preferably at least 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98% or 99% nucleotide sequence identity to any nucleotide sequence disclosed herein. In another example, a nucleic acid variant may hybridize to any nucleotide sequence described herein under high stringency conditions. A fragment may comprise or consist of up to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95-99% of the contiguous nucleotides present in any nucleotide sequence described herein. A fragment may comprise or consist of up to 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 950, 1000, 1050, 1100, 1150, 1200, 1350 or 1300 contiguous nucleotides present in any nucleotide sequence described herein.

[0127] The isolated nucleic acid(s) may be operably linked to one or more additional nucleotide sequences, such as one or more promoters and / or one or more enhancers. The one or more additional nucleotide sequences are typically regulatory nucleotide sequences. By operably linked or operably connected, it is meant that the regulatory nucleotide sequence(s) is / are positioned relative to the nucleic acid to be expressed such that it initiates, regulates or otherwise controls expression of the nucleic acid. As generally used herein, a "genetic construct" is an artificially created nucleic acid that incorporates and / or facilitates the use of the isolated nucleic acid disclosed herein. In certain instances, such constructs may be useful for recombinant manipulation, propagation, amplification, homologous recombination and / or expression of the isolated nucleic acid.

[0128] The regulatory nucleotide sequence will generally be appropriate for the host cell used for expression. For various host cells, numerous types of suitable expression vectors and suitable regulatory sequences are known in the art. The one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcriptional start and stop sequences, translational start and stop sequences, splice donor / acceptor sequences, and enhancer or activator sequences. Constitutive or inducible promoters known in the art may be used, including, for example, nisin-inducible, tetracycline-repressible, IPTG-inducible, alcohol-inducible, acid-inducible, and / or metal-inducible promoters. In one example, the expression vector includes a selection marker gene. The selection marker may be useful for the purpose of selection of transformed bacteria (e.g., bla, kanR, ermB, and tetR) or transformed mammalian cells (such as hygromycin, G418, and puromycin resistance).

[0129] Also described herein is a method for producing (i) a reporter protein of the invention, or (ii) a biosensor of the invention or a component thereof, comprising expressing the reporter protein, biosensor or component thereof in a suitable host cell. Host cells of the invention. Also described herein is a method for producing (i) a reporter protein of the invention, or (ii) a biosensor of the invention or a component thereof, comprising introducing one or more nucleic acids of the invention into a suitable host cell and incubating the host cell under conditions suitable for expression of the reporter protein, biosensor or component thereof. The method may be a recombinant expression method. As used herein, a genetic construct used for recombinant protein expression is referred to as an expression construct, in which the isolated nucleic acid to be expressed is operably linked or operably connected to one or more additional nucleotide sequences in an expression vector. An expression vector may be either a self-replicating extrachromosomal vector, such as a plasmid, or a vector that integrates into the host genome.

[0130] Suitable host cells for expression can be any of the following: prokaryotic or eukaryotic, for example bacterial cells including Escherichia coli (e.g., DH5α), yeast cells such as S. cerivisiae or Pichia pastoris, insect cells such as SF9 cells utilized in the baculovirus expression system, or a variety of mammalian or other animal host cells such as CHO, BHK or 293 cells. Introduction of the expression construct into a suitable host cell can be by techniques well known in the art, including, for example, electroporation, heat shock, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-based transfection (e.g., lipofectin, lipofectamine), protoplast fusion, microinjection, or particle bombardment.

[0131] Purification of recombinantly produced reporter proteins, biosensors or biosensor components can be performed by any method known in the art. In a preferred example, the recombinant protein molecule contains a fusion partner (preferably a C-terminal His tag) that allows purification by a suitable affinity matrix, which in the case of a His tag is a nickel matrix or resin. The resulting engineered variants are preferably expressed in bacteria such as E. coli as epitope-tagged proteins and purified by affinity chromatography.

[0132] The reporter protein or biosensor of the present invention may be expressed in a host cell, for example for metabolic engineering applications. This is shown, for example, in Figures 15 to 17. Suitable host cells in which the reporter protein and / or biosensor of the present invention may be expressed for metabolic engineering applications may be prokaryotic or eukaryotic. The host cell may be selected from microbial cells, bacterial cells (including, for example, Escherichia coli such as DH5α), yeast cells (for example, S.cerivisiae or Pichia pastoris), insect cells (such as SF9 cells utilized in a baculovirus expression system), plant cells (for example, wheat, maize or other crop cells), or mammalian cells (such as CHO, BHK or 293 cells). Preferably, the host cell is a microbial cell, a bacterial cell (for example, preferably an E.coli cell) or a yeast cell, most preferably a bacterial cell. Introduction of constructs for expressing the reporter proteins / biosensors described herein can be by any suitable technique known in the art, depending on the host cell, such as by electroporation, heat shock, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-based transfection (e.g., lipofectin, lipofectamine), protoplast fusion, microinjection or particle bombardment.

[0133] For example, reporter proteins / biosensors can be used to trigger or control certain properties of host cells in response to one or more target molecules. For example, a specific reporter protein / biosensor based on an antibiotic resistance marker (e.g., the reporter protein is β-lactamase or aminoglycoside phosphotransferase) may be used to construct a biosensor that induces antibiotic resistance in host cells, typically microbial cells (e.g., bacterial cells, yeast cells, E. coli cells, etc.), in response to one or more target molecules of the biosensor. For example, to select cells that produce high levels of a particular compound or protein of interest, a biosensor based on an antibiotic resistance marker is prepared, having the compound or protein of interest as a target molecule. The cells to be tested are mutagenized to express the biosensor, challenged with the relevant antibiotic, and surviving cell colonies are selected, including cells that produce enough of the target molecule to activate the biosensor and turn on antibiotic resistance enough to survive. See, for example, Figures 15-17. In such applications, the wide dynamic range of the biosensor of the present invention is very important, so that the host cell bacteria cannot survive by only producing more of the low activity resistant protein. Similarly, the reporter protein / biosensor can be based on fluorescence (e.g., FRET (fluorescence resonance energy transfer) system) or luminescence protein (e.g., BRET (bioluminescence resonance energy transfer) system) and can be used to construct a biosensor that induces fluorescence or luminescence in the host cell in response to one or more target molecules of the biosensor. For example, to select cells that produce high levels of a particular compound or protein of interest, a biosensor based on a fluorescent or luminescent protein is prepared, having the compound or protein of interest as a target molecule. The cells to be tested are mutagenized to express the biosensor(s), and the proximity of positivity can be detected by high levels of fluorescence / luminescence and can be isolated, for example, using fluorescence-activated cell sorting (FACS). The selected cells are those that produce enough of the target molecule to activate the biosensor and turn on high levels of fluorescence / luminescence.

[0134] Another application of the biosensor of the present invention is metabolic switching. For example, in certain applications, it is not desirable to produce a particular product until the host cells grow to an optimal or maximum density. A biosensor according to the present invention is prepared with a cellular metabolite as a target molecule, the production or high concentration of which indicates an optimal or maximum cell density. The biosensor is expressed in the host cell, and when the target metabolite reaches a desired level, the biosensor is activated with a reading or signal from the biosensor indicating that the cells are ready to be used to produce the desired product.

[0135] Further metabolic engineering applications will be apparent to those of skill in the art who, following the teachings of the present invention, can readily prepare appropriate reporter proteins and biosensors tailored to particular applications.

[0136] The invention may be better understood with reference to the following examples.

[0137] Aspects of the invention 1. A reporter protein comprising a first heterologous amino acid sequence responsive to binding of a first regulatory factor portion and a second heterologous amino acid sequence responsive to binding of a second regulatory factor portion, wherein binding of the first regulatory factor portion to the first heterologous amino acid sequence and binding of the second regulatory factor portion to the second heterologous amino acid sequence reversibly modulates the activity of the reporter protein.

[0138] 2. The reporter protein according to aspect 1, wherein the reporter protein is an enzyme and the activity of the reporter protein is the catalytic activity of the enzyme; or, the reporter protein is a fluorescent protein and the activity of the reporter protein is the fluorescence of the fluorescent protein.

[0139] 3. The reporter protein of embodiment 1 or 2, wherein binding of the first regulator portion to the first heterologous amino acid sequence and binding of the second regulator portion to the second heterologous amino acid sequence reversibly activates the activity of the reporter protein.

[0140] 4. The reporter protein according to any one of aspects 1 to 3, wherein the dynamic range of the reporter protein is at least 10-fold, optionally at least 20-fold.

[0141] 5. The reporter protein of any one of aspects 1-4, wherein the first heterologous amino acid sequence is provided as an insertion within the amino acid sequence of the reporter protein and the second heterologous amino acid sequence is provided as an insertion within the amino acid sequence of the reporter protein.

[0142] 6. The reporter protein according to any one of aspects 1 to 5, wherein the first heterologous amino acid sequence and the second heterologous amino acid sequence are inserted at different positions within the amino acid sequence of the reporter protein.

[0143] 7. The reporter protein according to any one of aspects 1 to 6, wherein the first heterologous amino acid sequence and / or the second heterologous amino acid sequence is a protein.

[0144] 8. The reporter protein according to any one of aspects 1 to 7, wherein the first heterologous amino acid sequence and the second heterologous amino acid sequence are proteins capable of undergoing a conformational change in response to binding of a regulatory moiety.

[0145] 9. The reporter protein according to any one of aspects 1 to 8, wherein the first heterologous amino acid sequence and the second heterologous amino acid sequence are the same protein or a functional fragment thereof.

[0146] 10. The reporter protein according to any one of aspects 1 to 9, wherein the first heterologous amino acid sequence and / or the second heterologous amino acid sequence is a calmodulin protein or an affinity clamp, or a functional fragment thereof.

[0147] 11. The reporter protein according to any one of aspects 1 to 10, wherein the reporter protein further comprises one or more additional heterologous amino acid sequences.

[0148] 12. The reporter protein according to any one of aspects 1 to 11, wherein the one or more further heterologous amino acid sequences are provided as an insertion within the amino acid sequence of the reporter protein.

[0149] 13. The reporter protein according to any one of aspects 1 to 12, wherein each heterologous amino acid sequence is inserted within the amino acid sequence of the reporter protein at a different insertion position.

[0150] 14. The reporter protein of any one of aspects 1 to 13, wherein binding of the regulatory portion to the first heterologous amino acid sequence causes a conformational change in the structure of the first heterologous amino acid sequence, and binding of the regulatory portion to the second heterologous amino acid sequence causes a conformational change in the structure of the second heterologous amino acid sequence.

[0151] 15. The reporter protein according to any one of aspects 1 to 14, wherein the first regulator portion and / or the second regulator portion is a peptide.

[0152] 16. The reporter protein according to any one of aspects 1 to 15, wherein the first regulator portion and the second regulator portion are the same peptide.

[0153] 17. A reporter protein according to any one of aspects 1 to 16, wherein the first regulator moiety and / or the second regulator moiety is a calmodulin-binding peptide or an affinity clamp RGS peptide ligand.

[0154] 18. A reporter protein according to any one of aspects 1 to 17, wherein the first regulatory factor portion and / or the second regulatory factor portion is a calmodulin-binding peptide having reduced binding affinity for a calmodulin protein compared to a wild-type calmodulin-binding peptide.

[0155] 19. The reporter protein of embodiment 18, wherein the first and second regulator portions are linked together, optionally via a linker, optionally as a fusion protein.

[0156] 20. The reporter protein of embodiment 19, wherein the first and second regulatory moieties are each further linked to a protein which is a variant of the first or second heterologous amino acid sequence, respectively, that has a reduced binding affinity for the regulatory moiety compared to the binding affinity of the respective heterologous amino acid sequence on the reporter protein.

[0157] 21. The reporter protein of embodiment 20, wherein (i) the first and second heterologous amino acid sequences are calmodulin proteins and (ii) the first and second regulator moieties are calmodulin-binding peptides, each further linked to a calmodulin protein having reduced binding affinity for the calmodulin-binding peptide compared to the binding affinity of the calmodulin protein on the reporter protein.

[0158] 22. A reporter protein according to any one of aspects 1 to 21, wherein activity of the reporter protein is activated upon binding of the calmodulin-binding peptide to first and second heterologous amino acid sequences, both of which are calmodulin.

[0159] 23. The reporter protein according to any one of aspects 1 to 22, wherein (i) the reporter protein is an enzyme selected from the group consisting of trehalase, oxidoreductase, glucose dehydrogenase, β-lactamase, aminoglycoside phosphotransferase, α-amylase, and carbonic anhydrase; or (ii) the reporter protein is a fluorescent protein selected from the group consisting of GFP, Cherry, mNeon, bacterial phytochrome (BphP)-based fluorescent protein, or cyanobacteriochrome (CBCR)-derived fluorescent protein.

[0160] 24. The reporter protein according to any one of aspects 1-23, wherein the reporter protein further comprises a first binding moiety B1'.

[0161] 25. The reporter protein according to any one of aspects 1-24, wherein the first and second regulator moieties are linked together and further linked to a binding moiety B1″ capable of interacting with a first binding moiety B1′ on the reporter protein.

[0162] 26. The reporter protein of embodiment 25, wherein the first and second regulator moieties and the binding moiety B1″ are linked as a fusion protein, optionally via one or more linkers.

[0163] 27. The reporter protein of embodiment 25, wherein the interaction between the binding moieties B1' and B1" causes the first regulator moiety, the second regulator moiety, and the reporter protein to co-localize, thereby enhancing binding of the first and second regulator moieties to the first and second heterologous amino acid sequences, respectively, thereby activating activity of the reporter protein.

[0164] 28. The reporter protein according to any one of aspects 1-24, wherein the reporter protein further comprises a second binding moiety B2'.

[0165] 29. A reporter protein according to any one of aspects 1-28, wherein the first regulator portion comprises a binding moiety B1″ capable of interacting with a first binding moiety B1′ on the reporter protein.

[0166] 30. The reporter protein of embodiment 28 or 29, wherein the second regulator portion comprises a binding moiety B2'' capable of interacting with a second binding moiety B2' on the reporter protein.

[0167] 31. The reporter protein according to any one of aspects 28 to 30, wherein the interaction of binding moieties B1' and B1" and the interaction of binding moieties B2' and B2" modulates and optionally activates the activity of the reporter protein.

[0168] 32. A reporter protein according to any one of aspects 28 to 31, wherein the activity of the reporter protein is activated only upon both the interaction of binding moieties B1' and B1" and the interaction of binding moieties B2' and B2" .

[0169] 33. The reporter protein according to any one of aspects 24 to 32, wherein the interaction between the binding moieties B1' and B1'' co-localizes the first regulator moiety and the reporter protein, thereby enhancing binding of the first regulator moiety to the first heterologous amino acid sequence.

[0170] 34. The reporter protein according to any one of aspects 28 to 33, wherein the interaction between binding moieties B2' and B2'' co-localizes the second regulator moiety and the reporter protein, thereby enhancing binding of the second regulator moiety to the second heterologous amino acid sequence.

[0171] 35. The reporter protein according to any one of aspects 28 to 34, wherein the interaction between binding moieties B1' and B1'', and between binding moieties B2' and B2'', co-localize the first regulator moiety, the second regulator moiety, and the reporter protein, thereby enhancing binding of the first and second regulator moieties to the first and second heterologous amino acid sequences, respectively, thereby activating activity of the reporter protein.

[0172] 36. A reporter protein according to any one of aspects 24 to 35, wherein binding moieties B1' and B1'' are capable of direct binding to each other and / or binding moieties B2' and B2'' are capable of direct binding to each other.

[0173] 37. A reporter protein according to any one of aspects 24 to 36, wherein the interaction of the binding moieties B1' and B1'' depends on the presence of the first target molecule TM1.

[0174] 38. A reporter protein according to any one of aspects 28 to 37, wherein the interaction of the binding moieties B2' and B2'' depends on the presence of a second target molecule TM2.

[0175] 39. A reporter protein according to any one of aspects 28 to 38, wherein the interaction of binding moieties B1' and B1'' and the interaction of binding moieties B2' and B2'' depend on the presence of both a first target molecule TM1 and a second target molecule TM2, such that activity of the reporter protein is activated only in the presence of both target molecules TM1 and TM2.

[0176] 40. A reporter protein according to any one of aspects 24 to 39, wherein in the presence of a first target molecule TM1, a first binding moiety B1' on the reporter protein interacts with a binding moiety B1'' on a first regulator moiety.

[0177] 41. A reporter protein according to any one of aspects 28 to 40, wherein in the presence of a second target molecule TM2, a second binding moiety B2' on the reporter protein interacts with a binding moiety B2'' on the second regulator moiety.

[0178] 42. A reporter protein according to any one of aspects 24 to 41, wherein binding moieties B1' and B1'' form a binding moiety pair that simultaneously binds to the target molecule TM1.

[0179] 43. A reporter protein according to any one of aspects 28 to 42, wherein the binding moieties B2' and B2'' form a binding moiety pair that simultaneously binds to the target molecule TM2.

[0180] 44. A reporter protein according to any one of aspects 28 to 43, wherein the binding moieties B1' and B1'' and the binding moieties B2' and B2'' are the same binding moiety pair or different binding moiety pairs.

[0181] 45. A reporter protein according to any one of aspects 37 to 44, wherein the target molecules TM1 and TM2 are the same or different.

[0182] 46. ​​The reporter protein according to any one of aspects 37 to 45, wherein in the presence of the first target molecule TM1 and the second target molecule TM2, the first binding moiety B1' on the reporter protein interacts with the binding moiety B1'' on the first regulator moiety and the second binding moiety B2' on the reporter protein interacts with the binding moiety B2'' on the second regulator moiety, such co-localization of the reporter protein and the first and second regulator moieties results in binding of the first regulator moiety to the first heterologous amino acid sequence on the reporter protein and binding of the second regulator moiety to the second heterologous amino acid sequence on the reporter protein, thereby reversibly modulating and optionally activating the activity of the reporter protein.

[0183] 47. A reporter protein according to any one of aspects 1 to 46, wherein the presence of the first target molecule TM1 and the second target molecule TM2 reversibly regulates the activity of the reporter protein and optionally activates the activity of the reporter protein.

[0184] 48. (a) a reporter protein according to any one of embodiments 1 to 47; (b) a first regulatory portion as defined in any of embodiments 1 to 47; and (c) a second regulatory element portion as defined in any one of embodiments 1 to 47. A biosensor comprising:

[0185] 49. (a) a reporter protein according to any one of embodiments 1 to 47; or (b) a biosensor according to embodiment 48. A composition or kit comprising:

[0186] 50. A biosensor according to embodiment 48 or a composition or kit according to embodiment 49, wherein the reporter protein is an enzyme and further comprises a substrate for the enzyme.

[0187] 51. A biosensor, composition or kit according to any one of aspects 48 to 50, further comprising a target molecule TM1.

[0188] 52. The biosensor, composition or kit according to any one of aspects 48 to 51, further comprising a target molecule TM2.

[0189] 53. The biosensor, composition or kit according to any one of aspects 48 to 52, wherein the target molecule TM1 is the same as the target molecule TM2; or the target molecule TM1 is different from the target molecule TM2.

[0190] 54. The biosensor, composition or kit according to any one of aspects 48 to 53, further comprising a biological sample, which may or may not comprise the target molecule TM1 and / or the target molecule TM2.

[0191] 55. The biosensor, composition or kit according to any one of aspects 48 to 54, wherein the reporter protein is an enzyme and further comprises a second enzyme comprising a heterologous amino acid sequence that is responsive to peptide P, and wherein binding of peptide P to the heterologous amino acid sequence reversibly modulates the catalytic activity of the second enzyme, and a substrate for the second enzyme is the catalytic product of an enzyme according to aspects 1 to 47.

[0192] 56. The biosensor, composition or kit according to embodiment 55, wherein (a) the second enzyme is an oxidoreductase, optionally glucose dehydrogenase; (b) the heterologous amino acid sequence is calmodulin or a functional fragment thereof; and (c) the peptide P is a calmodulin-binding peptide.

[0193] 57. A method for detecting one or more target molecules, comprising contacting a reporter protein according to any one of embodiments 1 to 47; a biosensor or a composition or kit according to any one of embodiments 48 to 56 with a sample under conditions suitable for detecting the presence or absence of one or more target molecules in the sample.

[0194] 58. The method of embodiment 57, wherein the one or more target molecules comprise or consist essentially of target molecule TM1 and target molecule TM2.

[0195] 59. The method according to aspect 57 or 58, wherein the target molecule TM1 and the target molecule TM2 are the same or different.

[0196] 60. The method of any one of aspects 57-59, wherein the one or more targeting molecules are selected from the group consisting of methotrexate, phenol glucoside, rapamycin, tacrolimus, and cyclosporin A.

[0197] 61. A method for diagnosing a disease or condition in an organism, comprising contacting a reporter protein according to any one of aspects 1 to 47; a biosensor or a composition or kit according to any one of aspects 48 to 56 with a sample obtained from the organism under conditions suitable for detecting the presence or absence of one or more target molecules in the sample, wherein the presence or absence of the one or more target molecules in the sample indicates whether the organism has, or is at risk of having, the disease or condition.

[0198] 62. A method for monitoring one or more target molecules in an organism, comprising contacting a reporter protein according to any one of aspects 1 to 47; a biosensor or a composition or kit according to any one of aspects 48 to 56 with a sample obtained from the organism under conditions suitable for detecting and / or quantifying the presence or absence of one or more target molecules in the sample.

[0199] 63. The presence or absence of one or more target molecules in a sample indicates a particular physiological state in an organism, and optionally (i) the method is for monitoring a fertility cycle, and the one or more target molecules are hormones, such as progesterone, estradiol, luteinizing hormone (LH) and follicle stimulating hormone (FSH); (ii) the method is for monitoring levels of psychological stress, and the one or more target molecules is cortisol or α-amylase; (iii) the method is for monitoring a metabolic state, and the one or more target molecules are insulin and glucose; (iv) the method is for monitoring levels of a therapeutic drug, such as methotrexate, rapamycin, tacrolimus, or cyclosporine A; (v) the method is for monitoring levels of environmental contaminants, such as phenolic glucosides; or (vi) the method is for monitoring a metabolite of interest; The method according to embodiment 62.

[0200] 64. A method for monitoring a metabolite of interest in an organism, comprising expressing in the organism a reporter protein according to any one of aspects 1 to 47 or a biosensor according to any one of aspects 48 to 56 under conditions suitable for detecting and / or quantifying the metabolite of interest in the organism, optionally wherein the organism is a bacterial cell.

[0201] 65. A method for assaying for protein-protein or protein-small molecule interactions, comprising contacting a reporter protein according to any one of aspects 1 to 47; a biosensor or a composition or kit according to any one of aspects 48 to 56 with a sample under conditions suitable for detecting the presence or absence of an interaction between binding moieties B1' and B1" and / or an interaction between binding moieties B2' and B2"; or an interaction between binding moieties B1' and B1" and target molecule TM1, and / or an interaction between binding moieties B2' and B2" and target molecule TM2.

[0202] 66. The method of embodiment 65, wherein the binding moieties B1' and B1'' and the binding moieties B2' and B2'' are proteins.

[0203] 67. The method according to aspect 65 or 66, wherein the target molecule TM1 and the target molecule TM2 are proteins or small molecules.

[0204] 68. The method according to any one of aspects 65-67, wherein the binding moieties B1' and B1'' and the binding moieties B2' and B2'' are the same binding moiety pair, and the target molecule TM1 and the target molecule TM2 are the same.

[0205] 69. A detection device comprising a cell or chamber containing a reporter protein according to any one of embodiments 1 to 47 or a biosensor according to any one of embodiments 48 to 56.

[0206] 70. One or more nucleic acids encoding a reporter protein according to any one of embodiments 1 to 47 or a biosensor according to any one of embodiments 48 to 56.

[0207] 71. One or more expression vectors comprising one or more nucleic acids according to embodiment 70 operably linked to one or more promoters.

[0208] 72. A host cell comprising a reporter protein according to any one of aspects 1 to 47, a biosensor according to any one of aspects 48 to 56, one or more nucleic acids according to aspect 70, or one or more expression vectors according to aspect 71.

[0209] 73. A method for converting a constitutively active enzyme into an enzyme whose catalytic activity is reversibly regulated depending on the presence of one or more target molecules, comprising: (a) generating a library of single insertion enzyme mutants by inserting heterologous amino acid sequences responsive to binding of a regulatory factor moiety at several different positions within the enzyme sequence, and selecting single insertion enzyme mutants that exhibit an alteration in catalytic activity upon binding of the regulatory factor moiety; (b) generating a library of double-insertion enzyme mutants by inserting an additional heterologous amino acid sequence at a second site within the enzyme sequence that is responsive to binding of the regulator moiety, and selecting double-insertion enzyme mutants that exhibit altered catalytic activity in the presence of the regulator moiety and have an increased dynamic range compared to the corresponding single-insertion enzyme mutants. A method comprising:

[0210] 74. The method of embodiment 66, wherein the change in catalytic activity occurs in less than 10 minutes, optionally less than 5 minutes.

[0211] 75. A reporter protein according to any one of aspects 1 to 17, wherein the first regulator portion and the second regulator portion are the same calmodulin binding peptide (CaM-BP) and the first and second heterologous amino acid sequences of the reporter protein are the same calmodulin protein; and optionally the CaM-BP is further linked, optionally via a linker, to a calmodulin protein having reduced binding affinity for the CaM-BP compared to the calmodulin protein of the reporter protein.

[0212] 76. A reporter protein according to embodiment 18, wherein the linker connecting the calmodulin protein with reduced binding affinity and the CaM-BP comprises a protease cleavage site.

[0213] 77. A biosensor comprising a reporter protein of embodiment 75 or 76 and a first regulator portion and a second regulator portion as defined in embodiment 75 or 76 configured to detect a protease as a target molecule.

[0214] 78. A method for detecting the activity of a reporter protein according to any one of aspects 1 to 47, wherein the reporter protein is an enzyme and an electrochemical method is used, the electrochemical method being capable of distinguishing the enzyme substrate from the product; optionally, hydrolysis of the substrate is monitored electrochemically; optionally, the method is carried out in a biological fluid, such as serum; optionally, the reporter protein is β-lactamase and the substrate is nitrocefin. EXAMPLES

[0215] As alluded to in the introduction, the ensemble nature of allosteric systems poses a major problem in generating states where one conformation dominates the ensemble. The design of the following exemplary biosensors of the invention was based on the assumption that in the chimeric switch system, the regulatory domain allows for a specific array of conformations in each of its conformational states. It is expected that the active "on" state will likely be similar to that of the parent molecule, while the "off" state can be achieved by a wide range of mechanisms. Thus, much of the engineering challenge centers on creating an efficient and reversible "off" state in the reporter protein. As shown for the first time herein, conformation-induced inactivating changes in the reporter protein, created by two or more insertions of heterologous amino acid sequences at different sites, interact epistatically, resulting in the combined effects shown herein.

[0216] Example 1 – Trehalase-based AND-gated biosensor A series of insertion mutants of the enzymes trehalase and calmodulin were constructed. Trehalase breaks down trehalose to glucose, and its activity can be easily monitored by a coupled glucose oxidation reaction. The trehalase structure (PDB:2jg0) was analyzed and eight trehalase-calmodulin (Tre-CaM) chimeras were designed. The regulatory calmodulin domain was placed at a position in the enzyme sequence where it was hypothesized that the insertion was unlikely to perturb the overall folding or active site of the enzyme. The resulting chimeric proteins were expressed in Escherichia coli (E. coli) and purified to homogeneity. The activity of the chimeric proteins was assayed using a coupled assay in which trehalase oxidizes the generated glucose to gluconolactone by PQQ-glucose dehydrogenase (GDH). The assay was optimized such that the activity of trehalase is rate-limiting and therefore appears as a first-order reaction. Using this assay, the activity of wild-type trehalase as well as Tre-CaM chimeras was monitored in the presence or absence of a calmodulin-binding peptide (CaM-BP). As can be seen in Figure 2D, some mutants showed little or no activity, some were active but showed no CaM-BP dependency, and some were activated to different degrees by the peptide. The switchable variants showed a moderate dynamic range of 2- to 11-fold, mainly due to incomplete inactivation of the reporter in the absence of the ligand peptide. Analysis of selected variants confirmed that, as expected, they bound to CaM-BP with nanomolar affinity.

[0217] To test for additive effects, chimeric enzymes were constructed combining multiple insertions identified in the primary screen. For this purpose, an insertion at position 104 was combined with an insertion at either position 321 or position 440 (Figure 3A). These chimeras exhibited a dynamic range of over 50-fold and bound CaM-BP in a saturable and dose-dependent manner (Figures 3B, 3E), resulting in an apparent Kd of 68 nM. This is likely an overestimate since the fitting procedure does not take into account the dual binding site (Figure 3F).

[0218] We tested the ability of this 2CaM-Tre switch to operate in the context of a larger biosensor system. Accordingly, the N- and C-termini of 2CaM-Tre were fused to an FKBP domain that can form a ternary complex with the target molecule rapamycin and the FRB domain, respectively (Figure 4A). When the FKBP-2CaM-Tre-FKBP fusion protein was mixed with a fusion of FRB with the low-affinity CaM-BP developed in our previous study [4], the resulting system showed rapamycin-dependent activity and a large dynamic range (Figure 4B, C). This experiment demonstrates that the developed AND-gated reporter protein construct can be incorporated into a higher-order biosensor signaling system.

[0219] Further biosensors were constructed to test whether the individual CaM proteins included in the 2CaM-Tre switch could each be operated with different inputs. To this end, a variant biosensor was constructed in which a cyclophilin (CPY) domain was attached to the C-terminus of the FKBP-2CaM-Tre fusion to generate the FKBP-2CaM-Tre-CPY fusion protein. Such a construct was able to associate with FRB-CaM-BP in a rapamycin-dependent manner and with a fusion polypeptide containing calcineurin A and B (CalA / B) and CaM-BP (CalA / B-CaM-BP) in a cyclosporine A-dependent manner (Figure 4D). As shown in Figure 4E, addition of either of the ligands rapamycin or cyclosporine A to a mixture of FKBP-2CaM-Tre-CPY, FRB-CaM-BP, and Cal A / B-CaM-BP led to a mild activation of trehalase activity. However, addition of both ligands simultaneously led to full activation of the system (Figure 4D,E).

[0220] Example 2 – GDH-based AND-gate biosensor A series of insertion mutants of the enzymes glucose dehydrogenase (GDH) and calmodulin were constructed. GDH catalyzes the conversion of glucose to gluconolactone with the production of electrons. The activity of GDH was monitored using 5-methylphenazinium methylsulfate (PMS) as the electron mediator and 2,6-dichlorophenolindophenol (DCPIP) as the reporter dye, which changed from blue to colorless upon reduction. The GDH structure (PDB:1c9u) was analyzed and a library of CaM domain insertions was generated. The regulatory calmodulin domain was placed at a position within the enzyme sequence where the insertion was hypothesized to be unlikely to disrupt the overall folding of the enzyme. The resulting chimeric proteins were expressed in Escherichia coli (E. coli) and purified to homogeneity. The activity of the chimeric proteins was assayed using the DCPIP assay. The activity of wild-type GDH and GDH-CaM chimeras was monitored in the presence or absence of CaM-BP (M13). The single insertion mutants GDH-CaM_48N and GDH-CaM_212N exhibited CaM-BP-dependent activity with rapid activation times and moderate dynamic ranges of about 4- or 5-fold, respectively (FIG. 11A,B). A chimeric enzyme was constructed that combined a first CaM insertion at position 48 with a second CaM insertion at position 212. As shown in FIG. 11C, this chimera exhibited a more than 20-fold enhanced dynamic range, bound CaM-BP in a saturable and dose-dependent manner, and exhibited clinically useful activation times.

[0221] Example 3 – β-lactamase-based AND-gate biosensor BLA-CaM A series of insertion mutants of the enzymes β-lactamase (BLA) and calmodulin were constructed. β-lactamase breaks the β-lactam ring in nitrocefin, which can be detected by a color change from yellow to red (Figure 5A). The β-lactamase structure (PDB: 3gmw) was analyzed and a series of calmodulin (BLA-CaM) chimeras were designed. The regulatory calmodulin domain was placed again at a position in the enzyme sequence where the insertion was hypothesized to be unlikely to disrupt the overall folding of the enzyme (Figure 5B). The resulting chimeric proteins were expressed in Escherichia coli (E. coli) and purified to homogeneity. The activity of the chimeric proteins was assayed using the nitrocefin assay. The activity of wild-type β-lactamase and the BLA-CaM chimeras was monitored in the presence or absence of CaM-BP (M13). Twenty (20) different insertion sites were analyzed, as shown in Figure 5C. For the construction of dual switch variants, we selected single insertion mutants BLA-CaM 41G and BLA-CaM 197E, which exhibited CaM-BP-dependent activity with rapid activation times and a moderate dynamic range of approximately 7- or 8-fold, respectively (Figure 6A-D). A chimeric enzyme was constructed combining a first CaM insertion at position 41 with a second CaM insertion at position 197 (Figure 6E). As shown in Figures 5F and 5G, this chimera exhibited a remarkable dynamic range of more than 7000-fold, bound CaM-BP in a saturable and dose-dependent manner, and exhibited clinically useful activation times.

[0222] Analysis of the catalytic activity of the developed switch modules demonstrates only a moderate decrease in activity of the double-insertion chimera compared to the parent single-switch enzyme. The double-insertion variant retained approximately 25% of the activity observed for the wild-type enzyme (Figure 6H). Importantly, the double-insertion mutant exhibited significantly higher maximal activity compared to the best-performing single variant (CaM-BLA 253G chimera), which retained less than 5% of the maximal activity compared to the wild-type enzyme (Figure 6I).

[0223] FKBP-2CaM-BLA-FKBP & FRB-CaM-BP To test the utility of the developed 2CaM-BLA switch module, we supplied the FKBP domain at both the N- and C-termini (FKBP-2CaM-BLA-FKBP) and tested its activity in the presence or absence of rapamycin, as well as the fusion between the FRB domain and a truncated calmodulin-binding peptide (FRB-CaM-BP) (Figure 7A). Addition of rapamycin to solutions of FKBP-2CaM-BLA-FKBP and FRB-CaM-BP increased β-lactamase activity in a dose-dependent manner (Figure 7B). Fitting of the data revealed an apparent K of 11 nM. d and a dynamic range of 171-fold was obtained (Figure 7C). The biosensor was fully operational within 10 min (Figure 7D).

[0224] FKBP-2CaM-BLA-FKBP and calcineurin A / B-CaM-BP The FRB domain was then replaced with a fusion of calcineurin A and B to form the calcineurin A / B-CaM-BP fusion (Figure 8A) and the response of the mixture of FKBP-2CaM-BLA-FKBP and calcineurin A / B-CaM-BP to tacrolimus was tested. Addition of tacrolimus to the mixture increased β-lactamase activity in a dose-dependent manner (Figure 8B). Fitting of the data revealed an apparent K of 14 nM. d and a 60-fold dynamic range was obtained (Figure 8C). The biosensor was fully operational within 10 min (Figure 8D).

[0225] MTX biosensor (VHH-2CaM-BLA-VHH&nanoCLAMP-CaM-BP) To test the developed biosensor platform for small molecule analytes with different structures, we constructed a biosensor for methotrexate (MTX) by fusing 2CaM-BLA to a VHH binder for methotrexate (PDB: 3qxv). A nanoCLAMP domain capable of recognizing the VHH:MTX complex was then fused to a low affinity calmodulin-binding peptide. Addition of MTX to a mixture of VHH-2CaM-BLA-VHH and nanoCLAMP-CaM-BP rapidly increased β-lactamase activity (Figure 9B), achieving a comparable maximum activity when VHH-2CaM-BLA-VHH was activated by addition of the M13-binding peptide. The response was dose-dependent (Figure 9C), with an apparent K d The value was 7.3 nM, which is close to the affinity of the VHH:MTX interaction (Figure 9D). The biosensor achieved half-maximal activation in 5 min and full activation in 15 min (Figure 9E-F).

[0226] The utility of the developed test was validated by porting the assay onto a Beckman AU-480 clinical chemistry analyzer (Figure 10A), and the VHH-2CaM-BLA-VHH-based assay demonstrated the ability to quantitate MTX in human serum (Figure 10B,C).

[0227] Thermostable MTX biosensor To test that the MTX biosensor platform can operate across a variety of β-lactamase-calmodulin variant sequences, a thermostable β-lactamase-calmodulin chimeric MTX biosensor was constructed. The biosensor was constructed by constructing a 2CaM-BLA insertion mutant (SEQ ID NO: 86) from a thermostable BLA containing K55Q, S82A, G92D, T140K, H153R, V184A mutations (TEM-1 BLA, SEQ ID NO: 85). The use of the mutant TEM-1 BLA resulted in increased thermostability (Figure 22B). This 2CaM-BLA was then fused to a VHH binder for methotrexate (PDB: 3qxv) to generate a thermostable VHH-2CaM-BLA-VHH biosensor (SEQ ID NO: 87, also called 2VHH 2CaM-BLA-41G 197E). We then fused the nanoCLAMP domain, capable of recognizing the VHH:MTX complex, to a low-affinity calmodulin-binding peptide.Addition of MTX to a mixture of VHH-2CaM-BLA-VHH and nanoCLAMP-CaM-BP rapidly increased β-lactamase activity (Figure 2F).

[0228] Example 4 - Signaling cascade containing AND-gated biosensors The presented approach can be applied to multiple enzymes, allowing the modulation of enzymatic and signaling cascades.

[0229] Trehalose to glucose with input of rapamycin; glucose to gluconolactone with input of cyclosporin A To illustrate the application of biosensors in such a system, the two-input switch module shown in Figure 4F was combined with a CaM-GDH biosensor responsive to cyclosporine A, as previously described [4] (Figure 4G). In this system, the production of glucose from trehalose by 2FKBP-Tre-2CaM and FRB-CaM-BP biosensors is regulated by an input of rapamycin. The subsequent conversion of glucose to gluconolactone with the production of electrons by CaM-GDH-CpY and Cal A / B-CaM-BP biosensors is controlled by cyclosporine A, which regulates GDH activity. Activity analysis of such a system revealed that rapamycin and cyclosporine A individually produced near-background responses, but their combination led to a large increase in activity, consistent with the idea of ​​pathway regulation at multiple nodes.

[0230] Trehalose to glucose with input of rapamycin; glucose to gluconolactone with input of MTX To illustrate the application of multiple two-input switches in a cascade, the two-input switch shown in FIG. 4F was combined with a CaM-GDH biosensor responsive to MTX (FIG. 21A). In this system, the production of glucose from trehalose by FKBP-2CaM-Tre-CpY and FRB-CaM-BP biosensors is regulated by an input of rapamycin. The subsequent conversion of glucose to gluconolactone with the production of electrons by 2CaM-GDH-2VHH (also referred to as VHH-2CaM-GDH-VHH) and nanoCLAMP-CaM-BP biosensors is controlled by MTX, which regulates GDH activity. Activity analysis of such a system revealed that rapamycin and MTX individually produced near-background responses, but their combination led to a large increase in activity (FIG. 21B), consistent with the idea of ​​pathway regulation at multiple nodes.

[0231] References JPEG2025502052000002.jpg102158 Sequence Listing

[0232] SEQ ID NO:1 - Trehalase [ka]

[0233] SEQ ID NO:2 - with C-terminal linker and 6His tag Trehalase [ka]

[0234] SEQ ID NO:3 - β-lactamase [ka]

[0235] SEQ ID NO:4 - with C-terminal linker and 6His tag β-lactamase [ka]

[0236] SEQ ID NO:5-PQQ-GDH [ka]

[0237] SEQ ID NO:6 - with C-terminal 6His tag PQQ-GDH [ka]

[0238] SEQ ID NO:8 - Aminoglycoside phosphotransferase [ka]

[0239] SEQ ID NO:9 - with N-terminal linker and 6His tag Aminoglycoside phosphotransferase [ka]

[0240] SEQ ID NO:10 - Carbonic anhydrase [ka]

[0241] SEQ ID NO:11 - Exemplary calmodulin protein [ka]

[0242] SEQ ID NO: 12 - An exemplary sequence with an N-terminal linker and a C-terminal linker Calmodulin protein [ka]

[0243] SEQ ID NO:13-Linker1-GSGG

[0244] SEQ ID NO:14-Linker2-GGSSG

[0245] SEQ ID NO:15 - Exemplary wild-type calmodulin protein [ka]

[0246] SEQ ID NO:16 - Calmodulin protein with reduced binding affinity to wild-type CaM-BP [ka]

[0247] SEQ ID NO:17 - Wild type CaM-BP (M13 CaM-BP) [ka]

[0248] SEQ ID NO:18-CaM-BP 高親和性 [ka]

[0249] SEQ ID NO:19 -CaM-BP 低親和性 [ka]

[0250] Variant CaM-BP sequence: [ka]

[0251] SEQ ID NO:36-FKBP [ka]

[0252] SEQ ID NO:37-FRB [ka]

[0253] SEQ ID NO:38 - Calcineurin alpha subunit [ka]

[0254] SEQ ID NO:39 - Calcineurin beta subunit [ka]

[0255] SEQ ID NO: 40-CalA / B( Linker ) [ka]

[0256] SEQ ID NO:41 - Human serum albumin (HAS) GA binder [ka]

[0257] SEQ ID NO:42 - Cyclophilin [ka]

[0258] SEQ ID NO: 43 - α-amylase binding antibody VHH1 [ka]

[0259] SEQ ID NO: 44 - α-amylase binding antibody VHH2 [ka]

[0260] SEQ ID NO:45 - HAS antibody VHH binder [ka]

[0261] SEQ ID NO:46 - Methotrexate binding antibody VHH [ka]

[0262] SEQ ID NO:47 - nanoCLAMP (for methotrexate biosensor) [ka]

[0263] SEQ ID NO:48 - Includes N- and C-terminal linker sequences Affinity clamping [ka]

[0264] SEQ ID NO:49-SH3 [ka]

[0265] SEQ ID NO:50-SH3L [ka]

[0266] SEQ ID NO:51-2 CaM - Tre G104_P321 [ka]

[0267] SEQ ID NO:52-2 CaM - Tre G104_S440 [ka]

[0268] SEQ ID NO:53-2 FKBP - Tre -2 CaM (Figure 4A) [ka]

[0269] SEQ ID NO:54- FKBP - Tre - 2CaM - Cyclophilin (Figure 4D) [ka]

[0270] SEQ ID NO:55- BLA - CaM 41G (Figure 6A) [ka]

[0271] SEQ ID NO:56- BLA - CaM 197E (Figure 6C) [ka]

[0272] SEQ ID NO:57-2 CaM - BLA (BLA-CaM 41G_197E) (Figure 6E, F) [ka]

[0273] SEQ ID NO:58- BLA - 2CaM - 2FKBP (Figs. 7A and 8A) [ka]

[0274] SEQ ID NO:59- VHH - BLA - 2CaM 41G 197E- VHH (Figure 9A) [ka]

[0275] SEQ ID NO:60- GDH - CaM 48N (Figure 11A) [ka]

[0276] SEQ ID NO:61- GDH - CaM 212N (Figure 11B) [ka]

[0277] SEQ ID NO:62-2 CaM - GDH (GDH-2CaM48N_212N) (Figure 11C) [ka]

[0278] SEQ ID NO:63- PQQ-GDH - CaM Fusion (adapter enzyme) [ka]

[0279] SEQ ID NO:64- CaM - GDH - Cyclophilin (Figure 4G) (Adapter enzyme) [ka]

[0280] SEQ ID NO:65- FRB - CaM-BP 高親和性 (Figs. 4A and 7B) [ka]

[0281] SEQ ID NO:66- SUMO - CalA / B - CaM-BP 高親和性 (Fig. 4D, G, Fig. 8B) [ka]

[0282] SEQ ID NO:67- nanoCLAMP - CaM-BP [ka]

[0283] SEQ ID NO:68 - Modified pACYDuet BLA-CaM253G (vector used to express BLA in DH5α cells) [ka] [ka] [ka]

[0284] SEQ ID NO:69- Signal peptide - BLA - CaM253G [ka]

[0285] SEQ ID NO:70- Signal peptide - BLA - CaM253G - CaM-BP [ka]

[0286] SEQ ID NO:71 - pACYDuet modified-BLA-2CaM 41G 197E vector [ka] [ka] [ka]

[0287] SEQ ID NO:72 - pET30modified-BLA-2CaM 41G 197E vector [ka] [ka] [ka]

[0288] SEQ ID NO:73 - mNeon [ka]

[0289] SEQ ID NO:74- mNeon - CaM [ka]

[0290] SEQ ID NO:75- mNeon -2 CaM [ka]

[0291] SEQ ID NO:76-Affinity clamp [ka]

[0292] SEQ ID NO:77-Linker1-EDAPESG

[0293] SEQ ID NO:78- eGFP - GDH [ka]

[0294] SEQ ID NO:79- eGFP - GDH - Affinity clamping -54 [ka]

[0295] SEQ ID NO:80- eGFP - GDH - Affinity clamping -46 [ka]

[0296] SEQ ID NO:81- eGFP - GDH - Affinity clamping -46-54 [ka]

[0297] SEQ ID NO:82 - RGS peptide-ligand of GDH-affinity clamp [ka]

[0298] SEQ ID NO:83 - α-Amylase [ka]

[0299] SEQ ID NO:84 2VHH -2 CaM - GDH (Fig.21) [ka]

[0300] SEQ ID NO:85 Thermostabilized TEM-1 b-lactamase 253G K55Q, S82A, G92D, T140K, H153R, V184A mutants (Figure 22) [ka]

[0301] SEQ ID NO: 86 Thermostabilized β-lactamase-calmodulin chimera 253G K55Q, S82A, G92D, T140K, H153R, V184A (Figure 22): 2CaM - BLA-41G 197E Mutants (K55Q, S82A, G92D, T140K, H153R, V184A) [ka]

[0302] SEQ ID NO: 87 Thermostable β-lactamase-calmodulin chimera 253G K55Q, S82A, G92D, T140K, H153R, V184A fused to two VHH domains (Figure 22): 2CaM - BLA-41G 197E - 2VHH Mutants (K55Q, S82A, G92D, T140K, H153R, V184A) [ka]

[0303] SEQ ID NO: 88 nanoCLAMP-CaM-BP fusion with optimized affinity for ligand-dependent activation of SEQ ID NO: 84. (FIG. 21). [ka]

[0304] SEQ ID NO:89-2 CaM - GDH (GDH-2CaM 48N_212N) (variant of SEQ ID NO: 62) [ka]

Claims

1. A reporter protein comprising a first heterologous amino acid sequence responsive to binding of a first regulatory factor portion and a second heterologous amino acid sequence responsive to binding of a second regulatory factor portion, wherein binding of the first regulatory factor portion to the first heterologous amino acid sequence and binding of the second regulatory factor portion to the second heterologous amino acid sequence reversibly modulates the activity of the reporter protein.

2. the reporter protein is an enzyme and the activity of the reporter protein is the catalytic activity of the enzyme; or the reporter protein is a fluorescent protein and the activity of the reporter protein is the fluorescence of the fluorescent protein; and / or 2. The reporter protein of claim 1, wherein the dynamic range of the reporter protein is at least 10-fold, and optionally at least 20-fold. (a) a first heterologous amino acid sequence is provided as an insertion within the amino acid sequence of the reporter protein, and a second heterologous amino acid sequence is provided as an insertion within the amino acid sequence of the reporter protein; and / or (b) the first heterologous amino acid sequence and / or the second heterologous amino acid sequence is a protein; and / or (c) the first heterologous amino acid sequence and the second heterologous amino acid sequence are proteins capable of undergoing a conformational change in response to binding of a modulator moiety; and / or (d) the first heterologous amino acid sequence and the second heterologous amino acid sequence are the same protein or functional fragment thereof; and / or (e) the first heterologous amino acid sequence and / or the second heterologous amino acid sequence is a calmodulin protein or a functional fragment thereof; A reporter protein according to claim 1 or 2.

4. (a) the first regulator moiety and / or the second regulator moiety is a peptide; (b) the first modulator portion and the second modulator portion are the same peptide; and / or (c) the first regulator moiety and / or the second regulator moiety is a calmodulin-binding peptide; and / or (d) binding of the regulator portion to the first heterologous amino acid sequence causes a conformational change in the structure of the first heterologous amino acid sequence, and binding of the regulator portion to the second heterologous amino acid sequence causes a conformational change in the structure of the second heterologous amino acid sequence; optionally, binding of the calmodulin-binding peptide to the first and second heterologous amino acid sequences, both of which are calmodulin, activates the activity of the reporter protein; and / or (e) The reporter protein according to claim 1 or 2, wherein (i) the reporter protein is an enzyme selected from the group consisting of trehalase, oxidoreductase, glucose dehydrogenase, β-lactamase, aminoglycoside phosphotransferase, α-amylase, and carbonic anhydrase; or (ii) the reporter protein is a fluorescent protein selected from the group consisting of GFP, Cherry, bacterial phytochrome (BphP)-based fluorescent protein, or cyanobacteriochrome (CBCR)-derived fluorescent protein.

5. The reporter protein further comprises a first binding moiety B1', and optionally further comprises a second binding moiety B2'; and / or (a) the first and second regulator portions comprise a binding portion B1″ that is capable of interacting with a first binding portion B1′ on the reporter protein; or (b) the first modulator portion comprises a binding portion B1″ that is capable of interacting with a first binding portion B1′ on the reporter protein; or (c) the second modulator portion comprises a binding portion B2″ capable of interacting with a second binding portion B2′ on the reporter protein; or (d) the first regulator portion comprises a binding moiety B1" capable of interacting with a first binding moiety B1' on the reporter protein, and the second regulator portion comprises a binding moiety B2" capable of interacting with a second binding moiety B2' on the reporter protein; A reporter protein according to claim 1 or 2.

6. (A) (a) the interaction of binding moieties B1′ and B1″ modulates the activity of the reporter protein, and optionally activates the activity of the reporter protein; or (b) the interaction between binding moieties B1′ and B1″ and between binding moieties B2′ and B2″ modulates the activity of the reporter protein, and optionally activates the activity of the reporter protein; or (c) the activity of the reporter protein is activated only upon both the interaction of binding moieties B1′ and B1″ and the interaction of binding moieties B2′ and B2″; and / or (B) (a) binding moieties B1' and B1" can be directly bonded to each other, and / or binding moieties B2' and B2" can be directly bonded to each other; or (b) the interaction of binding moieties B1' and B1" is dependent on the presence of a first target molecule TM1; and / or the interaction of binding moieties B2' and B2" is dependent on the presence of a second target molecule TM2, optionally where target molecules TM1 and TM2 are the same or different; and / or (C) the interaction of binding moieties B1' and B1" and the interaction of binding moieties B2' and B2" are dependent on the presence of both a first target molecule TM1 and a second target molecule TM2, such that the activity of the reporter protein is activated only in the presence of both target molecules TM1 and TM2, optionally the target molecules TM1 and TM2 being the same or different; and / or (D) binding moieties B1' and B1" and binding moieties B2' and B2" are the same binding moiety pair or different binding moiety pairs; and / or (E) The reporter protein of claim 5, wherein the presence of the first target molecule TM1 and the second target molecule TM2 reversibly regulates the activity of the reporter protein and optionally activates the activity of the reporter protein.

7. (a) the reporter protein of claim 1; (b) a first regulator moiety as defined in claim 1; and (c) a second regulatory element moiety as defined in claim 1. A biosensor comprising:

8. (a) the reporter protein of claim 1 A composition or kit comprising:

9. (b) A biosensor according to claim 7. A composition or kit comprising:

10. (a) the reporter protein is an enzyme and further comprises a substrate for the enzyme; and / or (b) further comprising a target molecule TM1; and / or further comprising a target molecule TM2; optionally, the target molecule TM1 is the same as the target molecule TM2; or the target molecule TM1 is different from the target molecule TM2; and / or (c) further comprising a biological sample, which may or may not comprise target molecule TM1 and / or target molecule TM2; and / or (d) the reporter protein is an enzyme and further comprises a second enzyme comprising a heterologous amino acid sequence responsive to peptide P, wherein binding of peptide P to the heterologous amino acid sequence reversibly modulates the catalytic activity of the second enzyme, and the substrate of the second enzyme is the catalytic product of the enzyme of claim 1; optionally, (a) the second enzyme is an oxidoreductase, optionally a glucose dehydrogenase, (b) the heterologous amino acid sequence is calmodulin or a functional fragment thereof, and (c) peptide P is a calmodulin-binding peptide. A biosensor according to claim 7 or a composition or kit according to claim 8 or 9.

11. 10. A method for detecting one or more target molecules, comprising contacting a reporter protein according to claim 1; a biosensor according to claim 7; or a composition or kit according to claim 8 or 9; with a sample under conditions suitable for detecting the presence or absence of one or more target molecules in the sample; Depending on the situation, (a) the one or more target molecules comprise or consist essentially of target molecule TM1 and target molecule TM2; and / or (b) the target molecule TM1 and the target molecule TM2 are the same or different; and / or (c) the one or more targeting molecules are selected from the group consisting of methotrexate, phenol glucoside, rapamycin, tacrolimus, and cyclosporin A; method.

12. (a) a method for diagnosing a disease or condition in an organism, comprising contacting a reporter protein according to claim 1; a biosensor according to claim 7 or a composition or kit according to claim 8 or 9 with a sample obtained from the organism under conditions suitable for detecting the presence or absence of one or more target molecules in the sample, wherein the presence or absence of the one or more target molecules in the sample indicates whether the organism has or is at risk of having the disease or condition; or (b) a method for monitoring one or more target molecules in an organism, the method comprising contacting a reporter protein according to claim 1, a biosensor according to claim 7 or a composition or kit according to claim 8 or 9 with a sample obtained from the organism under conditions suitable for detecting and / or quantifying the presence or absence of one or more target molecules in the sample; or (c) a method for monitoring a metabolite of interest in an organism, comprising expressing in the organism a reporter protein according to claim 1 or a biosensor according to claim 7 under conditions suitable for detecting and / or quantifying the metabolite of interest in the organism, optionally wherein the organism is a bacterial cell; or (d) A method of assaying for protein-protein or protein-small molecule interactions, comprising contacting a reporter protein according to claim 1; a biosensor according to claim 7; or a composition or kit according to claim 8 or 9; with a sample under conditions suitable for detecting the presence or absence of an interaction between binding moieties B1' and B1" and / or an interaction between binding moieties B2' and B2"; or an interaction between binding moieties B1' and B1" and target molecule TM1 and / or an interaction between binding moieties B2' and B2" and target molecule TM2.

13. A detection device comprising a cell or chamber containing the reporter protein of claim 1 or the biosensor of claim 7.

14. One or more nucleic acids encoding the reporter protein of claim 1 or the biosensor of claim 7; or one or more expression vectors comprising one or more nucleic acids operably linked to one or more promoters.

15. A host cell comprising the reporter protein of claim 1 or the biosensor of claim 7.

16. A host cell comprising one or more nucleic acids or one or more expression vectors described in claim 14.

17. 1. A method for converting a constitutively active enzyme into a reversibly regulated enzyme whose catalytic activity is dependent on the presence of one or more target molecules, comprising: (a) generating a library of single insertion enzyme variants by inserting heterologous amino acid sequences responsive to binding of a regulatory factor moiety at several different positions within the enzyme sequence, and selecting single insertion enzyme variants that exhibit altered catalytic activity upon binding of the regulatory factor moiety; (b) generating a library of double-insertion enzyme mutants by inserting an additional heterologous amino acid sequence at a second site within the enzyme sequence that is responsive to binding of the regulator moiety, and selecting those double-insertion enzyme mutants that exhibit altered catalytic activity in the presence of the regulator moiety and have an increased dynamic range compared to the corresponding single-insertion enzyme mutants; Including, In some cases, the change in catalytic activity occurs within less than 10 minutes, in some cases within less than 5 minutes; method.

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