Tunable biosensors and uses thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

TUNABLE BIOSENSORS AND USES THEREOFCROSS-REFERENCE

[0001] This application claims priority to U.S. provisional patent application no. 63 / 623,040, filed January 19, 2024, the contents of which are incorporated herein in their entirety.BACKGROUND

[0002] Field of the invention

[0003] The invention relates to tunable chimeric biosensors, which may comprise two-component systems (TCSs) and uses thereof. The invention relates to methods for engineering such tunable biosensors and their applications as building blocks in synthetic circuits for biocomputing.

[0004] Discussion of the related art

[0005] Evolution has developed a great number of natural sensor proteins for diverse applications, such as sensing cell population using their own quorum sensing system (Lubkowicz et al., 2018), or detecting specific small molecules of interest (Daeffler et al., 2017). Although a large repertoire of protein sensors is available in nature, the signals detected by most of them are unknown. Repurposing naturally occurring protein sensors as chemically different compounds is challenging and requires sophisticated protein engineering efforts to account for structurally different conformational states in the presence or absence of the signal (Leaver-Fay et al., 2011).

[0006] Cellular reprogramming implies changing the code of the biocomputing system. Signaling pathways that naturally integrates extra- and intra-cellular proteins are key components and can be engineered to build complex information processing devices.

[0007] TCSs are pathways present in bacteria, archaea, and some eukaryotes, including fungi and plants (Wuichet et al., 2010). These signaling pathways confer the ability to perceive a diverse set of signals, such as nutrients, temperature, light, etc. (Kreil et al., 2010) and diverse cellular processes are regulated in response, including cell division or differentiation (Ausmees & Jacobs- Wagner, 2003). The sensor protein is usually a histidine kinase (HK) whose auto-kinase activity is triggered in a signal-dependent mechanism (Buschiazzo & Trajtenberg, 2019). Upon phosphorylation, the HK transfers the phosphoryl- moiety to an invariant aspartate (Asp) in the effector response regulator (RR). RR often acts as a transcription factor, typically dimerizing to bind to specific promoters on the DNA in aphosphorylation dependent mechanism, thereby initiating the adaptative response (Gao et al., 2019).

[0008] Most HKs behave in a signal dependent manner as a specific phosphatase of their cognate phospho-RR. The phosphatase activity is relevant to shape the dynamics of the system (Gao & Stock, 2017), and to avoid nonspecific crosstalk with other TCSs (Siryaporn & Goulian, 2008), since the RR could be phosphorylated by a surrogate HK or small phospho-donors present in the cell.

[0009] Overall, specific HK:RR recognition is essential for unequivocally linking a particular signal with a specific response. Allosteric regulation of the multiple enzymatic activities involved in signal transduction ensures efficient information transmission and processing.

[0010] There is an unmet need for a new generation of chimeric biosensors that can be tuned for increased sensitivity and specificity in transducing an input signal into a functional response, as well as a modular platform to design and engineer such biosensors.SUMMARY OF THE INVENTION

[0011] The present disclosure describes tunable chimeric biosensors, as well as methods of producing and using such tunable chimeric biosensors. The present disclosure also describes an expanded toolbox of sensor modules able to transduce an input signal (e.g., intracellular and extracellular), and effector modules able to switch between distinct conformations and functional status in response to the input signal. Such molecular toolbox allows the design of versatile biosensors whose sensor modules and effector modules can be swapped independently according to the structural requirements and the desired applications in a plug-and-play manner.

[0012] Further aspects of the present disclosure are provided by the subject matter of the following clauses.

[0013] The invention relates to a tunable chimeric biosensor that include a chimeric dimer composed of a first protomer and a second protomer, each protomer including a sensor module operably linked to a HK catalytic module. The sensor module is capable of recognizing an input signal. Each protomer comprises at least one heptad repeat. The first protomer dimerizes with the second protomer into a closed conformation associated with a functional status. At least one heptad repeat of the first protomer assembles with at least one heptad repeat of the second protomer to form at least one coiled-coil structure. The at least one coiled-coil structure is downstream of the sensor module. At least one mutation may beintroduced in the HK catalytic module and / or the sensor module and / or in the at least one heptad repeat of at least one of the protomer to adjust a trigger threshold. Such trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled- coil structure. Upon reaching the trigger threshold, the at least one shift favors at least one transient open conformation of the dimer which induce a change in the functional status of the tunable chimeric biosensor.

[0014] The invention also relates to a tunable biosensor that includes a dimer composed of a first protomer and a second protomer. Each protomer includes a sensor module operably linked to an effector module by a helical linker; the effector module having a small split effector fragment connected to a large split effector fragment by a flexible linker of at least 5 residues; a dimerization module can also precede the flexible linker; the sensor module capable of recognizing an input signal; and the small effector fragment and large effector fragment capable of self-assembling into a functional module.

[0015] In an embodiment, the present disclosure relates to a tunable luciferase biosensor that includes a dimer composed of a first protomer and a second protomer, each protomer including a sensor module operably linked to a luciferase module by a helical linker, which includes a small split luciferase fragment (SmBit) connected to a large split luciferase fragment (LgBit) by a dimerization and histidine phosphotransfer (DHp) domain and a flexible linker of at least 5 residues. The sensor module is capable of recognizing an input signal and the SmBit and the LgBit are capable of self-assembling into a functional luciferase module. The helical linker comprises at least one coiled-coil structure extending from the sensor module and includes the SmBit fragment. The coiled-coil structure comprises at least one heptad repeat. In a closed conformation, the first protomer dimerizes with the second protomer, thereby precluding SmBit and LgBit from self-assembling in absence of the input signal. Upon recognizing the input signal, at least one allosteric change in the sensor module is transduced into at least one shift in the topology of the at least one coiled-coil structure which changes the topology of the coiled-coil structure and exposes SmBit, thereby allowing its assembly with LgBit into the functional luciferase module capable of emitting luminescence in the presence of a substrate.

[0016] At least one mutation may be introduced in the sensor module, the helical linker, or both, to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure. Upon reaching thetrigger threshold, the at least one shift favors a transient open conformation which changes the topology of the coiled-coil structure leading to the assembly SmBit:LgBit.

[0017] The invention also relates to a tunable DNA binding biosensor that includes a homodimer composed of two protomers, each protomer comprising a sensor module operably linked to a DNA binding domain. The sensor module is capable of recognizing an input signal. A helical linker comprising at least one coiled-coil structure extending from the sensor module operably links the sensor module to the DNA binding domain. The coiled-coil structure comprises at least one heptad repeat. The homodimer is configured in a closed conformation and binds to a target DNA sequence in absence of the input signal. At least one mutation may be introduced in the DNA binding domain or the sensor module, or the helical linker, or a combination of two or more of the DNA binding domains, the sensor module, and the helical linker, to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal requires to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure. Upon reaching the trigger threshold, the at least one shift favors a transient open conformation which disrupts the dimer interaction leading to the release of each protomer from the bound DNA target sequence.

[0018] The tunable biosensor of one of the preceding clauses, wherein the trigger threshold is decreased by introducing at least one mutation destabilizing the closed conformation to favor the transient open conformation which induces a change in the functional status of the tunable chimeric biosensor.

[0019] The tunable biosensor of one of the preceding clauses, wherein the trigger threshold is increased by introducing at least one mutation stabilizing the closed conformation associated with a functional status of the tunable chimeric biosensor.

[0020] As discussed more fully below with respect to the specific examples presented in Figures 5, 10 and 12, the invention relates to an in silico method for designing a tunable dimeric biosensor comprising a sensor module and an effector module. For example, method from Figure 5 involves generating a sensor module, the sensor module comprising two sensor domains, domain A and domain B, capable of assembling to form a ligand binding site in a closed conformation upon recognizing the ligand, including: identifying a ligand binding site, separating key residues of the ligand binding site into a first set of ligand binding residues and a second non-overlapping set of ligand binding residues. The first set of ligand binding residues and a helical segment are embedded within the domain B designed by partial diffusion or inpainting (Watson et al., 2023) to allow the folding of the first set of ligandbinding residues and the helical segment into a functional three-dimensional structure. The helical segment comprises at least one coiled-coil structure placed at the C-terminus of domain B, is positioned to favor the formation of a dimer in a closed conformation, and where the first set of the ligand binding residues is oriented towards the dimer interface upon dimerization. The second non-overlapping set of ligand binding residues is embedded in domain A designed by partial diffusion or inpainting to allow the folding of the second non- overlapping set of ligand binding residues into a functional three-dimensional conformation structure, where the first set of ligand binding residues and the second non-overlapping set of ligand binding residues form the functional ligand binding site capable of accommodating the ligand, and where domain A and domain B assemble into the functional sensor module capable of adopting an open or closed conformation. The sensor module is operably linked to the effector module through a helical linker including at least one heptad repeat.

[0021] The invention relates to a TCS that includes: a biosensor according to any of the preceding clauses that involves HK based biosensors, and a cognate RR comprising a receiver domain, wherein the activated biosensor transfers a phosphoryl moiety to a phosphorylation site of the cognate RR.

[0022] The invention relates to a biocomputing system that includes a plurality of TCSs according to the any of the preceding clauses that involves HK based biosensors, each TCS comprising a distinct biosensor and its cognate RR, wherein the plurality of TCSs can be connected through phosphorylation and / or dephosphorylation reactions to integrate and process information.DESCRIPTION OF THE DRAWINGS

[0023] Figs. 1A-L illustrate the biosensor general mechanism and the functionality of BioRapDesk integrating a sensor module formed by FKBP51 and FRB domains.

[0024] Figs. 2A-I show heptad repeat designs for biosensors using different HKs.

[0025] Figs. 3A-C show the process of tuning the activating threshold of biosensor by changing the energy landscape of the phosphatase inactive state.

[0026] Figs. 4A-C show the design and functionality of BioArgDesk, which integrates a ArtJ periplasmic binding protein as a sensor module.

[0027] Fig. 5 illustrates the design of split ligand binding domains.

[0028] Fig. 6 : Schematic illustrations of the different biosensor embodiments.

[0029] Figs. 7A-B show the design of a DNA binding biosensor.

[0030] Figs. 8A-C: Membrane biosensor design embodiments.

[0031] Figs. 9 is a schematic illustration of the pCOLA-Duet expression plasmid.

[0032] Figs. 10 is a schematic illustration of a macromolecular biosensor pipeline design.

[0033] Figs. 11A-B illustrates the design of macromolecular biosensors.

[0034] Figs. 12A-D illustrates the design of small ligand biosensors.

[0035] Figs. 13A-B illustrates the design of luciferase-based biosensor.

[0036] Figs. 14A-B illustrates the design of inverted biosensors.

[0037] Figs. 15A-D illustrates the design of kinetic models and biocomputing devices.DETAILED DESCRIPTION

[0038] In the last few years, protein engineering capabilities have been rapidly advancing thanks to the development of powerful computational algorithms and affordable DNA synthesis. Many efforts have focused on designing of customizable biosensors (Tinberg et al., 2013, Quijano-Rubio et al., 2021, Rihtar et al., 2023, Shui et al., 2021). The versatility of ligand recognition and its integration into information processing circuits are still not fully solved. TCSs were proposed as important tools for the construction of synthetic circuits two decades ago (Ninfa et al., 2007). However, the implementation of such signaling circuits is still limited.

[0039] The present embodiments relate to tailored-made and tunable chimeric biosensors that can be wired in TCS frameworks to construct biocomputing systems. In some embodiments, the tunable chimeric biosensors make use of phosphorylation reactions which allow rapid transmission and processing of information (Mishra et al. 2021). Since the unit of information transmission is a phosphoryl-moiety that derived from a molecule of ATP, the energetic cost is significantly reduced compared to information unit using a protein. Thus, the number of tunable chimeric biosensors that can be packed in a single cell is expected to be higher without exerting an energetic burden. Sophisticated circuits integrating the tunable chimeric biosensors of the embodiments are designed and integrated in novel hardware and software capable of implementing high-level programmed task for biological systems.

[0040] Some embodiments of the tunable chimeric biosensors, the methods and strategies for producing them, and their applications are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the invention is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent components can be used, andother methods developed without departing from the broad concepts of the current invention. All references cited anywhere in this specification, including the Background and Detailed Description sections, are incorporated by reference as if each had been individually incorporated.

[0041] Some definitions are included herein for the purpose of understanding the present subject matter and the appended claims. The abbreviations used herein have their conventional meanings within the chemical and biological arts.

[0042] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

[0043] The present description identifies certain amino acid sequences (peptides or polypeptides) as part of the invention. It is to be understood that the specifically identified sequences adequately describe other sequences that contain less than 100% sequence identity to the identified sequences but provide a similar function. For example, a polypeptide may contain less than 100% sequence identity to a polypeptide specifically identified herein while providing a similar function. For example, a polypeptide may have at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% sequence identity with a polypeptide specifically disclosed herein and still retain the same or sufficiently similar activity or functionality as the polypeptide specifically identified. In view of the present disclosure, persons skilled in the art will recognize that additional modifications, including sequence truncations, can be made to the proteins in non-determinant regions of the protein, without impacting function. Such modifications are disclosed as within the scope of the present disclosure.

[0044] As used throughout, the term “fragment” refers to a portion of an amino acid sequence which is identical in sequence to but shorter in length than a reference sequence. A fragment may comprise few amino acid residues and may not retain a function of the full protein or polypeptide it derived from. A fragment may comprise a truncated amino acid sequence of the protein or polypeptide while still retaining a specific function. In someembodiments, the split of a functional protein or a functional polypeptide may produce two fragments that upon reassembling produce a fully functional protein or polypeptide. For example, an enzyme capable of emitting light in the presence of a cognate substrate may be split into two non-functional fragments of different length that are unable to catalyze the substrate individually. Upon reassembly, both fragments complement each other and recreate the functional enzyme, restoring its ability to emit light in the presence of the substrate.

[0045] As used throughout, the term “histidine kinase (HK)” refers to a class of enzymes that share common 3D structures and catalytic mechanisms. The prototypical HK is an homodimeric protein comprising a sensor module, a transmission / transduction module, and a catalytic (CAT) module (Buschiazzo & Trajtenberg, 2019). The CAT module comprises the dimerization and histidine-phosphotransfer domain (DHp) and an ATP -binding domain (ABD). A HK binds ATP, transferring its y-phosphoryl moiety to a conserved histidine. The phosphorylated HK then transfers the phosphoryl to a conserved aspartate of its cognate response regulator. Non-limiting example of HKs include enzymes from HisKA, HisKA_3, HisKA_2 and HWE HK families.

[0046] As used throughout, the term “phosphorylation status” refers to the phosphorylation state of a protein. For example, if the HK catalytic module is in the phosphorylated or unphosphorylated state.

[0047] As used throughout, the term “trigger threshold” refers to the minimum amount of input signal required to transduce allosteric changes of the sensor module into shifts in the topology of the coiled-coil structure. Upon reaching the trigger threshold, an open conformation of the tunable chimeric biosensor of the invention is favored thereby inducing a change in its functional status. The trigger threshold can be tuned by introducing mutation(s), insertion(s) or deletion(s) of amino acid residues in any one or more domains or modules of the biosensor, i.e., the sensor module, the effector module such as the HK module or the helical linker.

[0048] As used throughout, the term “closed conformation” refers to the structural state in which the helical motif preceding the DHp domain forms a coiled-coil structure with the same motif of the other protomer of the dimeric structure.

[0049] As used throughout, the term “open conformation” refers to the structural state in which the helical motif preceding the DHp domain is not interacting in a coiled-coil arrangement with the motif of the other protomer of the dimeric structure.

[0050] As used throughout, the term “domain” refers to any region in a protein which can fold independently showing a particular structural feature.

[0051] As used throughout, the term “module” refers to a region (a motif, a domain, or a group of domains) that retains a functional property. In the context of the invention, a sensor module is responsible for sensing or detecting the presence, the concentration, or the presence and concentrations, of a target signal such as an analyte, a peptide, a protein or compound. The sensing module may comprise one or more domains. For example, a sensing module may comprise two split fragments of a ligand binding site of a protein which are capable of assembling upon binding to the ligand. A catalytic module is responsible for catalytic activities. For example, a catalytic domain of a HK biosensor may comprise at least one DHp domain, associated with dimerization, and one ABD domain, associated with ATP- binding.

[0052] A biosensor implies a signal dependent switch, triggering the transition between at least two conformations having different functional states. Thus, engineering a biosensor requires that more than one state simultaneously be taken into account during the design process.

[0053] In some embodiments, the tunable biosensor comprises a sensor module operably connected to an effector module, which activity is induced by conformational changes under the control of a coiled-coil switching mechanism. Non-limiting examples of effector modules are HK catalytic module, DNA binding module, luciferase module, enzymatic module or fluorescent protein module. For example, a HK biosensor can be designed based on the mechanistic understanding of the allosteric regulation of DesK, a thermosensor histidine kinase from Bacillus subtilis. starting from a phosphatase stabilized HK (Trajtenberg et al.. 2016, Saita et al.. 2015, Albanesi et al.. 2009, Lima et al.. 2023). Accordingly, the biosensor is constructed by connecting the catalytic and soluble portion of the HK with a novel sensor module at the N-terminus. The sensor module is operably linked to the HK module, for example by a helical linker comprising a coiled-coil structure, configured to favor the closed conformation of the HK. Further, to construct a homodimeric biosensor with a coiled-coil switch, a HK is connected at the C-terminus with a new sensor module at the N-terminus through a rigid helical linker. The sensor module is oriented and stabilized in a closed conformation through coiled-coil interactions between the protomers. The biosensor can be opened by the corresponding signal via a steric competition mechanism (Figure 1). In one aspect of the embodiment, a macromolecule biosensor (Figure 1; protomers in colors yellow and green) is stabilized in the closed conformation. The binding of the ligand (Figure 1; in color magenta) with the sensor module at the dimerization interface triggers the biosensor switch to the open conformation (Figure 1A). Further, interactions of the sensormodule with a peptide or a protein through the dimerization interface compete with the dimerized sensor module, displace the coiled-coil arrangement leading to a conformational switch that promotes the opening of the biosensor (Figure 1 A). In another aspect of the embodiment, small molecule biosensors involve a two-domain sensor module. The corresponding ligand binds to both sensor domains, triggering a conformational switching favoring the open conformation (Figure 1B). Further, the sensor module may comprise 2 distinct domains that assemble upon recognizing a ligand, resulting in the same topological change of the coiled-coil structure (Figure 1B). In another aspect of the embodiment, the effect(s) of the presence or absence of the ligands on the energetic state is disclosed (Figure 1C). In the ground state, which can be defined as the more stable configuration and higher populated state of the protein, the biosensor is stabilized in the closed conformation, whereas the open state is unfavorable (higher energy state) (Figure 1C). Upon ligand binding, the conformational equilibrium is shifted towards the open state by displacing the coiled-coil structure that connects the sensor domain with the catalytically active region of the HK (Figure 1C). Further, in the absence of the input signal, the coiled-coil structure is energetically stabilized in a packed closed conformation having, for example, a constitutive phosphatase state. In another embodiment, the closed conformation may be associated with a constitutive kinase active state. Such embodiment is possible when the phosphorylation site of the catalytic module is accessible in a closed conformation. Upon recognizing and binding the input signal, a conformational rearrangement occurs, an open conformation is favored, promoting the activation of the kinase competent state and the inactivation of the phosphatase state of the biosensor (Figure 1C). If the closed conformation is associated with an active kinase state, the conformational rearrangement leads to the inactivation of the kinase.

[0054] The biosensors of the invention show great versatility and allow activation to be triggered by an input signal such as by a small molecule, a peptide or a protein. Non- limiting examples of input signal may include heavy metals such as zinc or iron, small molecules such as lactate, adenosine or glucose. In another aspect of the embodiment, the biosensor strategy can be implemented in soluble or membrane configurations in order to sense intra- or extracellular signals without the need of signal internalization. An illustrative example of a biosensor kinase activated in the open conformation and kinase inactivated in the closed state is depicted in Figure 1D, where RR and the DNA are shown in blue and light blue colors, respectively. Further, a similar mechanism can be adapted for building soluble or membrane associated biosensors to detect intra or extracellular signals (Figure 1D).

[0055] Sensor module

[0056] A sensor module is responsible for sensing or detecting the presence, the concentration, or both the presence and the concentration of a target signal such as a small molecule, a peptide, a macromolecule or a physicochemical stimulus. In the context of the invention, the sensor module and the effector module are derived from different proteins or distinct naturally occurring biosensors, thereby resulting in a chimeric biosensor.

[0057] In some embodiments, the sensor module switches from an open conformation to a closed conformation upon binding to a ligand (domain A:ligand:domain B; as also discussed further in Figure 14 below). The perceived input signal induces structural rearrangements of the sensor module resulting in intramolecular conformational changes within the biosensor. In one aspect of the embodiments, structural rearrangements may trigger rotation, piston- or scissor-like movements, or a combination of such movements downstream of the biosensor. The mechanical switch from an open to a closed conformation and vice versa is possible because the sensor module is operably linked to the effector module by a coiled- coil structure. Non-limiting examples of sensor modules according to the embodiments are: i) BioRap (SEQ ID NO: 27), which recognizes rapamycin, is a fusion protein between FK506- binding protein 51 (FKBP51, SEQ ID NO: 25) and FKBP12-rapamycin binding protein (FRB, SEQ ID NO: 26); ii) BioArg (SEQ ID NO: 45), which recognizes arginine, derives from the periplasmic binding protein domain of ArtJ from the thermophilic bacterium Geobacillus stearothermophilus (e.g, SEQ ID NO: 43 and SEQ ID NO: 44); and iii) BioAba (e.g., SEQ ID NO: 49), which recognizes abscisic acid, is a fusion protein between Pyrabactin Resistance 1- Like 2 (PYL2, SEQ ID NO: 47) and Hypersensitive to ABA 1 (HAB1, e.g, SEQ ID NO: 48). In some embodiments, the sensor module comprises a first sensor domain (domain A) and a second sensor domain (domain B) capable of assembling upon recognizing the input signal. Figure 6 illustrates the general domain architecture of different biosensor embodiments. HK- based biosensors are illustrated in embodiments 1 through 12 in Figure 6. Arrows indicate protease cleavage sites (like SPAse I or II). SP denotes export signal peptide and TM transmembrane segments. Biosensors with different output effector motifs, like DNA binding domains (DBD), one or more domains with enzymatic activity, fluorescent reporters or domains with binding properties (split A and split B), are shown in embodiments 13 to 20 in Figure 6. Design embodiments 14 and 15 in Figure 6 include a marginally stable coiled-coil after the sensor module that promotes dimerization but can be destabilized by ligand binding. Biosensor design 16 in Figure 6 includes a dimerization module (DM, for example, a four-helix bundle like a DHp from a HK or other dimerizing proteins). In exemplary embodiments, weshow constructing a membrane bound biosensor, an export signal peptide with a protease cleavage site was included at the N-terminus and a transmembrane segment (TM) was inserted between the sensor module (BioArg or BioRap) and the HK (DesKC). The size of the TM and the length of the helical linker is adjusted to keep the relative orientation of sensor and catalytic modules (Figure 8A). E. coli cells transformed with a plasmid encoding a membrane biosensor was diluted to 0.1 OD600and incubated in the prescence of 10 μM IPTG and in absence (black) or presence (grey) of arginine (5 mM) in a plate reader (TEC AN 200 Pro) at 37 °C with agitation. Time courses of fluorescence levels ( λex= 475 nm, λem= 512 nm) normalized by OD450were registered over time. Data correspond to triplicates of a representative experiment (Figure 8B-C).

[0058] The first sensor domain may be connected to the second sensor domain by a flexible linker (Figure 6, embodiments 1, 4-6, and 13-16). Such linker provides more flexibility and accessibility. A linker may be a peptide rich in serine and / or glycine amino acid residues and include at least 5 amino acids, preferably 5 to 15 amino acids. In a preferred embodiment, the flexible linker comprises the synthetic sequences SEQ ID NO: 81 (GSSASGTSSTSSG) or SEQ ID NO: 20 (GGSGGGGSGGGSGGS). In other embodiments, the first sensor domain is not covalently linked to the second sensor domain (Figure 6, embodiments 2, 7-9).

[0059] In some embodiments, the first sensor domain and the second sensor domain each comprise split fragments of a protein that include a recognition site for the input signal. The split fragments may be of equal size, or one split fragment may be significantly shorter than the other split fragment. In one aspect of such embodiments, each split fragment is derived from a distinct portion of the protein. For example, the sensor module BioArg (SEQ ID NO: 45) is engineered from the substrate binding component of the ATP -binding cassette transport system ArtJ from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine (Vahedi-Faridi et al. 2008). The substrate binding component of ArtJ is folded into two distinct lobes with the interface between the lobes forming the substrate-binding pocket. The two lobes (e.g., SEQ ID NO: 43 and SEQ ID NO: 44) are used as domain A and domain B of the BioArg sensor module (SEQ ID NO: 45) and may be linked through a flexible linker as defined herein.

[0060] In other aspects of the embodiments, the first sensor domain and the second sensor domain are split fragments derived from distinct proteins but are nonetheless capable of forming a heterodimeric sensor module in the presence of the input signal or a ligand. Forexample, BioAba (e.g., SEQ ID NO: 49), the fusion protein between PYL2 (SEQ ID NO: 47) and HAB1 (SEQ ID NO: 48).

[0061] In other embodiments, the first sensor domain is absent, and the second sensor domain recognizes a macromolecule (Figure 6, embodiments 3, 10-12, 17-20). For example, BioFRBligDesk (SEQ ID NO: 100) is similar to BioRapDesk (SEQ ID NO: 29), but without the domain A. In such embodiment, the input signal is the macromolecule and its binding to the sensor module induces allosteric changes that transduce to the effector module.

[0062] Helical linker

[0063] As used throughout, the term “coiled-coil structure” is defined as a super- helical arrangement in which two or more alpha helices are wound around each other promoted by hydrophobic and polar interactions encoded in the periodic pattern of the protein sequence. Canonical coiled-coils comprise seven-residue sequence repeats or heptad repeats whose positions are labelled a-g with the core-forming positions (a and d) usually occupied by hydrophobic residues, while the remaining, (b, c, e, f, and g) are usually hydrophilic residues. The a and d residues of the heptad repeats of the helices interact via a knobs-into- holes geometry. Accordingly, a residue from one helix (knob) packs into a space surrounded by four sidechains of the facing helix (hole). Because the residue comes to be located next to the equivalent residue from the facing helix, this geometry is sometimes referred to as in- register. A mutation at a specific position of a heptad repeat, for example the substitution of a hydrophobic residue at the a and / or d position with a polar or hydrophilic residue may affect the stability of the coiled-coil structure.

[0064] In some embodiments, the sensor module and the HK module are operably linked by at least one coiled-coil structure which transduce conformational changes and modulate the transition among functional states of the tunable biosensor. For example, the activation of the tunable biosensor involves the disruption of the coiled-coil by helical rotation and / or lateral shift upon recognition of the input signal, favoring an open conformation that exposes the phosphorylatable His of the CAT domain, leading to an active auto-kinase state. Modifying the sequence of the helical linker and / or the heptad repeat affect the trigger threshold of the tunable biosensor.

[0065] In some aspects of the embodiments, at least one coiled-coil structure extends from the effector module to the sensor module through a helical linker. The helical linker is fused to the S-helix and may comprise one of the sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6 (LLEM), SEQ ID NO: 7 (LLEMLA), SEQ IDNO: 8 (LLEMLAK), SEQ ID NO: 9 (ILLEMLAK), SEQ ID NO: 10 (LLERLQSQD), SEQ ID NO: 11 (LLERLQSQAD), SEQ ID NO: 12 (LLEMMKQLEDKLA), SEQ ID NO: 13 (LLEMLAKMKQLEDKVARL), SEQ ID NO: 14 (LLEMLAK SRKERERLEEKLEDANER), SEQ ID NO: 15 (LLEMLAKSRKERERLEEKLEDANERIAE), SEQ ID NO: 16 (LLEMLAKSRKERERLEEKLEDANERIAEL), SEQ ID NO: 17 (LAKMKQLEDKVEELLSKNYHLENEVARL), SEQ ID NO: 18 (VKELEAEQKALEAE), SEQ ID NO: 19 (EKVLKELQANLKE); SEQ ID NO: 118 (NEYVKNNILLEMLA); SEQ ID NO: 121 (LLERLQAQD); SEQ ID NO: 122 (LEMVKELEAEQKALEA); SEQ ID NO: 123 (ALAKMKQAEDKVARLA); or SEQ ID NO: 124 (MKQLEDKVARL).

[0066] In some aspects of the embodiments, at least one coiled-coil structure comprises two or more heptad repeats. For example, a heptad repeat may deviate from the canonical sequence (abcdefg) with at least one of the core positions a and d occupied by a polar amino acid residue instead of a hydrophobic amino acid residue, the positions e or / and g being a polar or charged amino acid residue. Non-limiting examples of canonical linkers are LLEMLAKSRKERERLEEKLEDANERIAEL (SEQ ID NO: 16, canonical linker 1) or MKQLEDKVARL (SEQ ID NO: 124, canonical linker 2). The canonical sequence may be inserted upstream of the DHp domain. For example, BioRapNarx construct (SEQ ID NO: 52) comprises the canonical linker 1 (SEQ ID NO: 16), while several BioRapFix1 constructs (SEQ ID NO: 87) comprise the canonical linker 2 (SEQ ID NO: 124).

[0067] In an embodiment for the design of rapamycin biosensor, BioRapDesk contains FKBP51 domain covalently attached by a flexible linker to FRB domain in the N- terminus as the two-domain sensor module; a coiled-coil with heptad periodicity as connecting helical linker; and the catalytic and cytosolic region of DesK (DesKC) in the C- terminus as effector module. The rapamycin binding site of FRB domain faces the dimerization interface. In absence of rapamycin, the closed conformation (phosphatase state and kinase inactive state) is stabilized (Figure 1E). In some embodiments, the S-helix includes a stammer, approximately 4 helix turns N-terminally of the phosphorylatable histidine in the HK module. For example, a stammer is included in HK biosensor such as DesKC (SEQ ID NO: 28) or NarXC (SEQ ID NO: 51) of the HisKA_3 family (Figure 2A).

[0068] Transmembrane domain and signal peptide

[0069] In some embodiments, the tunable chimeric biosensor is in soluble form or membrane bound (Figure 6).

[0070] In some aspects of the invention, the tunable biosensor comprises at least one transmembrane domain (TD) (Figure 6, embodiments 4-6, 7 and 10). The transmembrane domain may be derived from a natural polypeptide or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein.

[0071] In some preferred embodiments, TD polypeptides preferentially comprise hydrophobic amino acids. For example, the TD comprises the amino acid sequenceSEQ ID NO: 1 (LLLLLLLLLLLLLLLLLLLLLL),SEQ ID NO: 2 (RLQSQDVALIVLLSTAIGLAGMAVSGWLV), SEQ ID NO: 3 (VALIVLLSTAIGLAGMAVSGWLV), SEQ ID NO: 4 (FWVWFFFLLSLGICVALVAFSSL), SEQ ID NO: 82 (LLLLLLLLALLLALLALLLALLLLLL), SEQ ID NO: 83 (ITLIIFGVMAIVIGTILLISYGIRRLIKKLA), SEQ ID NO: 84 (WWALLIFFATFAGFITFVFRFFW), or SEQ ID NO: 85 (IYLLIGLGLLSALLFLSWIVYL).

[0072] In some preferred embodiments, the amino acid sequence of the signal peptide comprisesSEQ ID NO: 21 (MKQSTIALALLPLLFTPVTKAAGSG),SEQ ID NO: 22 (MLKRCLSPLTLVNQVVLVHRVMAVFMALLLVFTIIWLSG), SEQ ID NO: 23 (MKKIWLALAGLVLAFSASAGGSG), SEQ ID NO: 24 (MRLRKYNKSLGWLSLFAGTVLLSGSG), or SEQ ID NO: 86 (MSKLKKIVIYLFFLCLGMHSAFSETP).

[0073] In some aspects of the embodiments, a TD is configured at the C-terminus of the second sensor module. The TD may be placed at the C-terminus of the helical linker such as in exemplary constructs mlBioRapDesk (SEQ ID NO: 65), m2BioRapDesk (SEQ ID NO: 66), m3BioRapDesk (SEQ ID NO: 67), m4BioRapDesk (SEQ ID NO: 68), m5BioRapDesk (SEQ ID NO: 69), m6BioRapDesk (SEQ ID NO: 70), m7BioRapDesk (SEQ ID NO: 71), m8BioRapDesk (SEQ ID NO: 72). In another aspect of the embodiments, a second TD or a signal peptide is configured either at the N-terminus or C-terminus of the sensor module such as in exemplary construct m2BioRapDesk (SEQ ID NO: 66) (Figure 6, embodiments 4-12). For periplasmic tunable biosensors, the sensor module may be framed by at least two TD and the HK catalytic module localized in the cytoplasm (Figure 6, embodiments 4-6).Accordingly, the sensory and kinase modules are located in different cellular compartments separated by a plasma membrane. In some embodiments, the TD may include amino acidresidues such as hydrophilic amino acids to promote intermembrane interactions which favor the closed conformation.

[0074] In other aspects of the embodiments, the second TD or the signal peptide is connected to the second sensor domain or the first sensor domain by a flexible linker. The combination of flexible and hydrophilic residues reduces the likelihood that the linker interferes with the folding and / or function of the domains. Such flexible linker may include a signal peptidase cleavage site (like a SPase I or II site) or a long segment of at least 10 amino acid residues (Figure 6, embodiments 4-12). Inserting a proteolytic cleavage downstream of the signal peptide prevents the use of long flexible linkers.

[0075] In other aspects of the embodiments, a functional membrane tunable biosensor comprising two TD recognizes an input signal in a different compartment or extracellular space. The first TD is configured at the N-terminus of the protomer followed by a flexible linker of at least 10 amino acid residues. The second TD is inserted between a sensor domain and the helical linker consisting of the S-helix. A peptidase cleavage site may be inserted immediately after the first TD, thus allowing for domain A and domain B to interact in the presence of the signal and trigger the switching mechanism. For example, different TD are used to generate membrane biosensors and evaluate domain folding and / or intramembrane interaction(s) and / or the triggering mechanism. Different types of signal peptides are selected to favor a co-translational transmembrane transport such as signal peptides derived from CyoA or DsbA as in m3BioRapDesk (SEQ ID NO: 67), m4BioRapDesk (SEQ ID NO: 68), m5BioRapDesk (SEQ ID NO: 69), m6BioRapDesk (SEQ ID NO: 70), m7BioRapDesk (SEQ ID NO: 71), m8BioRapDesk (SEQ ID NO: 72) or post-translational (from PhoA) and improve biosensor expression and folding in the periplasm.

[0076] In some preferred embodiments, the TD polypeptide sequence can be modified to facilitate the structural transition on the membrane. Non-limiting examples include the addition of at least one Leu residue at the N-terminus of the TD polypeptide. For example, SEQ ID NO: 1 (LLLLLLLLLLLLLLLLLLLLLL) was optimized to SEQ ID NO: 82 (LLLLLLLLALLLALLALLLALLLLLL).

[0077] Effector module

[0078] Histidine Kinase catalytic module

[0079] An embodiment directed to a tunable chimeric biosensor includes: a chimeric dimer composed of a first protomer and a second protomer, each protomer comprising a sensor module operably linked to a HK catalytic module. The sensor module is capable ofrecognizing an input signal. Each protomer includes at least one heptad repeat. The first protomer dimerizes with the second protomer into a closed conformation associated with a functional status. The at least one heptad repeat of the first protomer assembles with the at least one heptad repeat of the second protomer to form at least one coiled-coil structure. Downstream of the sensor module, at least one mutation is introduced in the HK catalytic module and / or the sensor module and / or in the at least one heptad repeat of at least one of the protomer to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure. Upon reaching the trigger threshold, the at least one shift favors at least one transient open conformation of the dimer which induce a change in the functional status of the tunable chimeric biosensor.

[0080] In some embodiments, the HK catalytic module includes a dimerization and DHp covalently connected to an ABD at the C-terminus. In many histidine kinases, the DHp includes the phosphorylatable His within coiled-coil structures comprising at least one heptad repeat. In one aspect of the embodiments, at least one additional coiled-coil structure protrudes outward from the DHp domain extending toward the sensor module. Such additional coiled-coil structures often have non-ideal coiled-coil heptad repeats comprising polar residues at critical positions a and / or d and heptad repeat register shifts. Non-ideal coiled-coil structures are less energetically stable, and as such are capable of switching in their topology. The marginally stable structure of the coiled-coil makes these effective molecular switches controlling the conformational rearrangements of the HK CAT module. An exemplary sequence for DHp comprises SEQ ID NO: 77.

[0081] An exemplary embodiment BioRapDesk (SEQ ID NO: 29) was constructed using the FKBP51 :rapamycin:FRB heterocomplex (Banaszynski et al., 2005) as a sensor module in such a way that the interaction between FKBP51 (SEQ ID NO: 25) and FRB (SEQ ID NO: 26), induced by rapamycin, displaces the FRB of and disrupts the coiled-coil structure. To build a phosphatase stabilized conformation, the FRB domain was fused at the N-terminus of the cytoplasmic and soluble portion of DesKC (SEQ ID NO: 28). DesK is a well-studied member of the HisKA_3 family from Bacillus subtilis that acts as a thermosensor regulating a fast homeostatic cell membrane response. The orientation of the FRB domain was designed to place the ligand binding site towards the other FRB of the second protomer in the dimeric structure of BioRapDesk and the sequence of the helical linker followed a hydrophobic pattern to promote coiled-coil interaction between the two protomers of the dimeric biosensor (Figure 1E). A flexible linker between FKBP51 and FRBcovalently connects both sensor domains. The flexible linker may comprise one of the sequences SEQ ID NO: 81 or SEQ ID NO: 20. The biosensor is stabilized in the close inactive phosphatase conformation by promoting the coiled-coil structure through sequence design of the helical linker. Thus, the active state should be unfavorable as shown in Figure 1C (higher associated energy state in the middle of the plot). In the presence of rapamycin, the heterocomplex FKBP51 : rapamycin ERB sterically displaces the dimeric interface at the level of FRB. Thus, ligand binding shifts the conformational equilibrium towards the active state by displacing the coiled-coil structure that connects the sensor domain with the catalytically active region of the HK. This mechanical switch favors the active state of the biosensor, promoting autophosphorylation and activation of the system. The coiled-coil machine strategy can be implemented in soluble or membrane biosensors to sense intra- or extra-cellular signals without the need of signal internalization (Figure 1D).

[0082] To validate the tunable biosensor design, the BioRapDesk biosensor (SEQ ID NO: 29; Figure 1E) was recombinantly expressed and purified. Using an ATP-NADH coupled assay, the autophosphorylation activity of the biosensor was tested in the presence or absence of the input signal, rapamycin. BioRapDesk was inactive in the absence of rapamycin and activated in the presence of the input signal (Figure 1F). Similarly, Phostag SDS-page assays indicate that BioRapDesk autophosphorylates in the presence of rapamycin, whereas very low activity is observed in its absence (Figure 1G-H). Using the Phostag approach, the degree of phosphorylation was measured after 15 min of autophosphorylation reaction at different rapamycin concentrations (Figure 1H), indicating an activating threshold of 1.5 μM. Altogether, the thermosensor Desk was successfully repurposed to a rapamycin biosensor using a strategy enabling the development of a highly versatile tool for synthetic biology applications.

[0083] In BioRap (SEQ ID NO: 27), the FRB is positioned to face the rapamycin binding site in the dimerization interface and stabilization of coiled-coil structure to favor the phosphatase state. Helical linkers between FRB (SEQ ID NO: 26) and DesKC (SEQ ID NO: 28) may comprise residues Leu258 and Leu262, corresponding to a and d positions within a heptad repeat and numeration according to BioRapDesk. Destabilizing mutation in BioRapDesk is exemplified in variants or BioRapDesk_d1 (SEQ ID NO: 41) or BioRapDesk_d2 (SEQ ID NO: 30) with the Ala265Val or Leu258Ser mutation at the d or a position of the heptad repeat, respectively. Biosensor variants having stabilizing mutations are BioRapDesk_s1 (SEQ ID NO: 32), BioRapDesk_s2 (SEQ ID NO: 34), BioRapDesk_s3 (SEQ ID NO: 36), BioRapDesk_s4 (SEQ ID NO: 37) and BioRapDesk_s5 (SEQ ID NO: 39).Non-limiting examples of stabilizing substitutions introduced in the HK module DesKC include Ser251Leu (SEQ ID NO: 33, SEQ ID NO: 35), Ser251Ala (SEQ ID NO: 38), Ser251Val (SEQ ID NO: 40), Ala265Leu (SEQ ID NO: 35), and the insertion of a full heptad repeat from GCN4 (SEQ ID NO: 80 MKQLEDK) in the helical linker as in construct BioRapDesk_s3 (SEQ ID NO: 36).

[0084] Some embodiments are directed to highly complex information processing networks comprising more than one tunable biosensor that can orthogonally recognize different RRs. To this end, a similar strategy is implemented to build different HK -based tunable biosensors comprising a sensor module such as BioRap operably linked to the catalytically active module of each HK by a coiled-coil register. Non-limiting examples of HKs include members of the HisKA_3, HisKA families or other HKs families (like HisKA_2 or HWE HK). To design functional HK-based tunable biosensors, differences in the energetic coupling between the sensor module and the catalytic modules must be considered. Accordingly, if the catalytic region of HK is intrinsically in a closed state, the biosensor construction should not further stabilize the coiled-coil structure. Destabilizing mutations should be introduced to allow switching to the open conformation. Conversely, for HK catalytic regions that are intrinsically open state, the design should include a few stabilizing mutations in the coiled-coil region to trap the kinase in the closed conformation. Non-limiting example of HK modules are derived from DesK, NarX, PhoQ, KinA or FixL. Preferably, the HK module is limited to the soluble and catalytically active region of HK such as DesKC (SEQ ID NO: 28), NarXC (SEQ ID NO: 51), KinAC (SEQ ID NO: 53), PhoQC1 (SEQ ID NO: 57), PhoQC2 (SEQ ID NO: 59) or FixLC (SEQ ID NO: 116). In an embodiment, the tunable biosensor BioRapNarx (SEQ ID NO: 52) was constructed by substituting DesKC module (SEQ ID NO: 28) of the biosensor BioRapDesk (SEQ ID NO: 29) with a NarXC module (SEQ ID NO: 51) (soluble and catalytically active region) using as a reference the coiled-coil discontinuity, which involves a stammer or a 4-residue deletion in the heptad repeat, upstream of the phosphorylatable histidine in the HisKA_3 family (Figure 2A).

[0085] Some embodiments disclose the autophosphorylation kinetics of BioRapDesk by ATP consumption using the ATP-NADH coupled assay by following NADH absorbance decrease at 340 nm, where BioRapDesk (20 μM) was incubated in the absence or presence of rapamycin (20 μM; Figure 1F, black and light grey lines, respectively) at pH 8, 25 °C. DesKC (Figure 1F; grey line) was partially active, consistent with previous work (Lima et al. 2023). Some embodiments disclose autophosphorylation kinetics of BioRapDesk followed by Phostag SDS-PAGE, where BioRapDesk (10 μM) was incubated with ATP (1 mM) andmagnesium chloride (5 mM) in the presence or absence of rapamycin (10 μM) at pH 8, 25 °C. The reactions were stopped at different times and analyzed by Phostag SDS-PAGE (Figure 1G). Some embodiments disclose rapamycin dose-response curve of BioRapDesk, where purified BioRapDesk was incubated with ATP (1 mM), magnesium chloride (5 mM) and varying concentrations of rapamycin (0-80 μM), at pH 8, 25 °C, until equilibria were reached (15 min) (Figure 1H). The degree of phosphorylation was determined by Phostag SDS-PAGE gels and densitometry (Figure 1H). A Hill equation was fitted to the data corresponding to three independent experiments (Figure 1H). Some embodiments disclose epifluorescence microscopy of E. coli cells expressing BioRapDesk and DesR (RR of DesK) in presence or absence of rapamycin (50 μM), where a biosensor system for cell assays was generated by constructing a plasmid encoding BioRapDesk and DesR in an operon regulated by a T51ac promoter (Figure 1I). E. coli TOP10F’ cells transformed with the biosensor plasmid showed rapamycin-dependent activation of fluorescence when revealed by microscopy in agarose pad (Figure 1I). Similar cellular assays analyzed through FACS showed high homogenous activation of the fluorescent reporter (Figure 1J). Transformed cells with the biosensor plasmid were diluted to 0.1 OD600and incubated with varying concentrations of rapamycin in a plate reader (TECAN 200 Pro) at 37 °C with agitation. Fluorescence ( λex= 475 nm, λem= 512 nm) time courses were registered and normalized by the number of cells ( OD450) over time (Figure 1K). Rapamycin dose-response curve of the in- cell biosensor system using the fluorescence / OD levels after 2 h is presented in Figure 1L. A Hill equation was fitted to normalized data from two independent experiments. A threshold of 1.5 μM was observed, since cultures incubated with >1.5 μM rapamycin showed detectable fluorescence (Figure 1L).

[0086] In other embodiments, KinAC (SEQ ID NO: 53), PhoQC1 (SEQ ID NO: 57), PhoQC2 (SEQ ID NO: 59) or FixLC (SEQ ID NO: 117), from the HisKA family were used to substitute DesKC from the tunable biosensor BioRapDesk and BioRapDesk variants (e.g., _d1, _d2, _s1_s2_s3_s4). PhoQ is a low Mg sensor from E. coli. The heptad repeat of HisKA family (like PhoQ, Kin A, CpxA, EnvZ or FixL) is different than that of the HisKA_3 family (like DesK or NarX). In some aspects of the embodiments, PhoQ and FixL biosensors were engineered by fusing the BioRap sensor module (SEQ ID NO: 27) to the PhoQC1 (SEQ ID NO: 57, soluble and cytoplasmic region of PhoQ), PhoQC2 (SEQ ID NO: 59) or FixLC (SEQ ID NO: 117 soluble and cytoplasmic region of FixL) modules, respectively (Figure 2G-I). In some other embodiments, the BioArg (SEQ ID: 45) or the BioAba (SEQ ID NO: 49) sensor module is fused to one of PhoQC1 or PhoQC2 effector modules. PhoQC may be stabilizedeither in a phosphatase or a kinase state when a GCN4 coiled-coil motif (SEQ ID NO: 80) is introduced at different positions (McClune et al.. 2019). Biosensors that are activated or inactivated can be constructed using a coiled-coil structure. Depending on the requirement, PhoQ biosensor variants are constructed to stabilize the closed conformation in the kinase active or kinase inactive states by incorporating different coiled-coil structures between the sensor module and the HK module.

[0087] In some embodiments, BioRapDesk variants with mutations at the helical coiled-coil linker were generated to weaken or strengthen the coiled-coil interactions, where the heptad repeat comprises at least one destabilizing mutation that destabilizes the coiled- coil structure, thereby modifying its energetic stability (Figure 3). Destabilizing the coiled- coil structure lowers the energetic barrier needed to switch from the closed conformation of the dimeric tunable biosensor toward a different functional state associated with an open conformation (Figure 3 A-C). For example, in Figure 3 A, protein sequence alignment shows destabilizing (d1 and d2) and stabilizing (s1) substitutions at the heptad repeat coiled-coil periodicity. In contrast, stabilizing mutations introduced in the heptad repeat registers that stabilize the coiled-coil increase the energetic barrier and shifts the conformational equilibrium toward the constitutive state associated with a closed conformation (Figure 3 A- C). For example, GCN4 coiled-coil motif stabilize the closed conformation of the tunable biosensor. Figure 3B shows a schematic representation of the proposed effect exerted in the energy landscape by mutations at the coiled-coil region. Based on the DesK-DesR kinetic model (Lima et al. 2023) and the coiled-coil switching mechanism (Albanesi et al. 2009, Saita et al. 2015, Trajtenberg et al. 2016), the energy landscape could be perturbed by substitutions that stabilize or destabilize functional states, making the biosensor tunable.Figure 3C shows rapamycin dose-response curves of BioRapDesk and its variants where data correspond to six independent experiments.

[0088] DNA binding module

[0089] An embodiment is directed to a tunable DNA binding biosensor that includes: a homodimer composed of two protomers, each protomer including a sensor module operably linked to a DNA binding domain by a helical linker. The sensor module is capable of recognizing an input signal. The helical linker includes at least one coiled-coil structure extending from the sensor module and linked to the DNA binding domain. The coiled-coil structure includes at least one heptad repeat. The homodimer of the tunable DNA binding biosensor is configured in a closed conformation and binds to a target DNA sequence inabsence of the input signal. At least one mutation is introduced in the DNA binding domain and / or the sensor module and / or the helical linker to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled- coil structure. Upon reaching the trigger threshold, the at least one shift favors a transient open conformation which disrupts the dimer interaction leading to the release of each protomer from the bound DNA target sequence. For example, a DNA biosensor BioRapDBD (SEQ ID NO: 74) is constructed by fusing the BioRap sensor module (SEQ ID NO: 27) and the helix-turn-helix (HTH) domain of DesR (Figure 7A-B). To construct a DNA binding domain biosensor (BioDBD), a sensor module was extended with a helical coiled-coil segment to promote dimerization, maintaining the relative orientation of the sensor modules. Upon ligand binding, the monomeric state is favored over the dimeric oligomerization state, releasing BioDBD from the DNA (Figure 7A). E. coli cells were transformed with a plasmid encoding a BioRapDBD (including the DNA binding domain of DesR) and a reporter GFP under the control of a synthetic promoter (derived from T5 promoter and including two DesR DNA binding sites placed in a specific position so that transcription is blocked upon BioRapDBD binding) (Figure 7B). Cultures were diluted to 0.1 OD600and incubated in the presence of increasing concentrations of IPTG in a plate reader (TECAN 200 Pro) at 37 °C with agitation. Fluorescence levels ( λex= 475 nm, λem= 512 nm) normalized by OD450were registered after 2 h. Note that higher levels of BioRapDBD (higher levels of IPTG) repressed the reporter gene, validating the design of the synthetic promoter and evidencing the requirement of dimerization to bind DNA. Data correspond to triplicates of a representative experiment (Figure 7B).

[0090] In some aspects of the embodiment, the effector module is a DNA binding domain that derived from a protein involved in the regulation of gene expression. Non- limiting examples of DNA binding domains are domains derived from transcription factors, like HTH domains from response regulators. In a preferred embodiment, DBD module comprises SEQ ID NO: 73.

[0091] In some aspects of the embodiment, a sensor module is operably linked to a DNA binding module by a helical linker comprising at least one heptad repeat. In absence of the sensor’s cognate ligand, the biosensor is in a homodimeric conformation with the DNA binding domain able to interact with a target nucleic acid sequence with high affinity. In the presence of the cognate ligand, domains A and B of the sensor module interact and compete with the dimerization promoted by the coiled-coil helical linker, destabilizing the homodimerand resulting in a monomeric state of the DNA binding module, thus preventing its interaction with the target DNA sequence. Further, in some embodiments, in the case of macromolecule biosensors, the sensor module can be formed only by domain B similar to split biosensors for proteins.

[0092] In some embodiments, the DBD module is fused to the sensor module BioArg (SEQ ID NO: 45) or BioAba (SEQ ID NO: 49) through a helical linker as defined herein.

[0093] In some aspects of the embodiments, the tunable DNA binding biosensor includes at least one mutation that is introduced in the DNA binding module and / or the sensor module and / or the helical linker to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure. Destabilizing mutations may include one or more substitutions selected from M251A, M251S, L254A and L254S. Stabilizing mutations may include the addition of one or more heptad repeats and / or combinations of stabilizing mutations. Stabilization by improving the coiled-coil packing (hydrophobic residues in a or d positions) increases the energetic barrier that governs the transition from a close coiled-coil structure (dimer) to an open (conformation). The mutations modulating the trigger threshold of the tunable DNA binding sensor are the same as the mutations introduced into the HK biosensor BioRapDesk (e.g., BioRapDesk_d1, BioRapDesk_d2, BioRapDesk_s1, BioRapDesk_s2, BioRapDesk_s3, BioRapDesk_s4, BioRapDesk_s5). The effect of stabilizing / destabilizing mutations should be equivalent to the HK derived biosensors.

[0094] Luciferase module

[0095] An embodiment directed to a tunable luciferase biosensor includes a dimer composed of a first protomer and a second protomer, each protomer comprising a sensor module operably linked to a luciferase module by a helical linker. The helical linker includes a small split luciferase fragment (SmBit, SEQ ID NO: 75) (Dixon et al., 2016). The helical linker is connected to the large split luciferase fragment (LgBit, SEQ ID NO: 76) (Dixon et al., 2016) by a flexible linker of at least 5 residues. The SmBit and the LgBit are capable of self-assembling into a functional luciferase module (Dixon et al., 2016). The sensor module is capable of recognizing an input signal. The helical linker includes at least one coiled-coil structure extending from the sensor module; the coiled-coil structure comprising at least one heptad repeat. The first protomer dimerizes with the second protomer into a closed conformation precluding SmBit and LgBit from self-assembling in absence of the inputsignal. The recognition of the input signal induces at least one allosteric change of the sensor module transduced into at least one shift in the topology of the at least one coiled-coil structure. Such shift favors at least one transient open conformation, exposing the SmBit motif, thereby allowing the self-assembling with LgBit into the functional luciferase module capable of emitting luminescence in the presence of a substrate.

[0096] In some aspects of the embodiments, the tunable luciferase biosensor includes at least one mutation that is introduced in the luciferase module and / or the sensor module and / or the helical linker to adjust a trigger threshold. The trigger threshold is the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure. Non- limiting examples of stabilizing mutations include E254A, E254V, E254L (according to BioRapLuc numeration) or any mutations that facilitate the coiled-coil packing. Non limiting examples of destabilizing mutations include L251A or L251S. Mutations are expected to generate changes in the energetic landscape, hindering or facilitating the switching mechanism from one state to the other, respectively.

[0097] In some aspects of the embodiments, the sensor module comprises a first sensor domain (domain A) and a second sensor domain (domain B) capable of assembling upon recognizing the input signal. The first sensor domain and the second sensor domain may be derived from distinct proteins. The first sensor domain may either be connected to the second sensor domain by a flexible linker or may not be covalently linked to the sensor second domain. The first sensor domain may be absent, and the second sensor domain recognizes a macromolecule. .

[0098] In an exemplary embodiment, the sensor module is BioRap (SEQ ID NO: 27) comprising the two sensor domains FKBP51 and FRB that are capable of assembling in the presence of rapamycin. In other exemplary embodiments, BioRap sensor module may be replaced by BioArg (SEQ ID NO: 45) (derived from periplasmic binding protein) and BioAba (SEQ ID NO: 49) (derived from a protein which comprises a substrate binding site that can be split into two separate domains capable or reassembling upon substrate binding) which sense arginine and abscisic acid, respectively.

[0099] Biosensors based on luciferase have applications in diagnostics for human, animal, or environmental health. For example, tunable luciferase biosensors may be used to build portable sensors for detecting small molecules, peptides, or macromolecules for a fast and easy readout. Further, different enzymes besides HKs can be accommodated as effector modules. For example, a split nanoluc luciferase (Luc) can be used, placing the SmBiTfragment as helical linker and the large LgBiT fragment at the C-terminus (Figure 13 A). The system is stabilized as a closed conformation. The signal triggers the conformational switch exposing SmBiT to LgBiT, reconstituting the luciferase activity (Figure 13 A). In the presence of oxygen and furimazine, the reconstituted luciferase catalyzes the formation of furimamide. This reaction releases energy in the form of light resulting in bioluminescence. Stabilizing and destabilizing mutations are expected to tune ligand response, as observed with BioRapdesk (Figure 3). In an exemplary embodiment, BioRapLuc (1 μM) was incubated with 1 x furimazine (Nano-Gio® Live Cell Assay System reagent, Promega) in the presence or absence of rapamycin (80 μM). After 15 min, the bioluminescence was measured using a LUMIstar OPTIMA luminometer (BMG LABTECH) (Figure 13B). Data correspond to triplicates of a representative experiment.

[0100] In an exemplary embodiment, the luciferase module derived from the enzyme nanoluc™ or luciferase (Figure 13A-B). In such embodiment, the enzyme is split into a short peptide of at least 11 residues (SmBit) and a longer fragment LgBit (Dixon et al., 2016). In a preferred embodiment, SmBit and LgBit are covalently linked by a DHp domain and flexible linker as described herein. In such embodiment, the split fragments reassemble only in the presence of an input signal. In a preferred embodiment, SmBit comprises the amino acid sequence set forth as SEQ ID NO: 75 and LgBit comprises the amino acid sequence set forth as SEQ ID NO: 76. An exemplary embodiment is BioRapDHpLuc (SEQ ID NO: 79).

[0101] In some embodiments, the tunable luciferase biosensor further includes additional dimerization domains, like DHp domain, to favor dimerization and improve the functionality of the luciferase biosensor. The sequence of DHp may comprise SEQ ID NO: 77. An exemplary embodiment is BioRapDHpLuc (SEQ ID NO: 79).

[0102] In some embodiments, a tunable fluorescent biosensor derived from splitFAST (Fluorescence-Activating and absorption-Shifting Tag) was designed. In such embodiment, the protein is split into a short peptide of at least 11 residues (CFAST11) and a longer fragment NFAST (Tebo et al. 2019). In such embodiment, the split fragments reassemble only in the presence of an input signal. In a preferred embodiment, CFAST11 comprises the amino acid sequence set forth as SEQ ID NO: 126 and NFAST comprises the amino acid sequence set forth as SEQ ID NO: 116. An exemplary embodiment is BioRapDHpFAST (SEQ ID NO: 96).

[0103] Plug-and-play platform

[0104] An embodiment is related to a modular platform for engineering tunable chimeric biosensors from an expanded toolbox of sensor modules capable of recognizing and transducing an input signal (e.g., intracellular, or extracellular), and effector modules capable of switching between closed and open conformations and functional status in response to the input signal. The toolbox of the invention allows the design of tunable chimeric biosensors whose sensor modules and effector modules can be swapped independently according to the structural requirements and the desired applications in a plug-and-play manner. Accordingly, the sensor module BioRap (SEQ ID NO: 27) is operably linked to a luciferase effector comprising fragments SmBit (SEQ ID NO: 75) and LgBit (SEQ ID NO: 76) through a helical linker comprising at least one heptad repeat, thereby creating a rapamycin dependent luciferase biosensor. The sensor module BioRap may be swapped with BioArg (SEQ ID NO: 45) or BioAba (SEQ ID NO: 49). Additional domains such as DHp (SEQ ID NO: 77) and / or one or more TD (as defined herein) may also be added to the design of the novel biosensor depending on its intended application. A signal peptide may also be added to the design. Notably, to modulate the sensitivity and specificity of the tunable biosensor, the toolbox is further expanded to variants of sensor modules and variants of effector modules. For example, one or more mutations is introduced in a heptad repeat of the effector module and / or helical linker to adjust the trigger threshold of the newly designed tunable biosensor (mutations as disclosed herein). To construct a biosensor with kinase activity in the closed conformation functional state, in an embodiment, BioRapPASDesk, the transmission module of the HK BvgS of Bordetella pertussis, including Linker- 1, a PAS domain and Linker-2, was inserted between the sensor module BioRap and the HK DesKC (Figure 14A). The transmission module implies a yin yang mechanism, namely, when Linker- 1 is in the closed conformation, Linker-2 is in the open state, and vice versa (Figure 14B) (Dupre et al. 2021). The inclusion of the transmission module inverts the switching mechanism of biosensors, generating a biosensor that is active in the dimeric closed conformation of the sensor module (Figure 14A). Transformed E. coli cells with BioRapDesk or BioRapPASDesk plasmids were diluted to 0.1 OD600and incubated in a plate reader (TEC AN 200 Pro) at 37 °C with agitation. After 1 h, fluorescence levels ( λex= 475 nm, λem= 512 nm), normalized by OD450, were registered. Data correspond to triplicates of a representative experiment (Figure 14B).

[0105] Modifying the energy landscape for tunability

[0106] The biosensors of the invention can be tuned by changing the energy landscape required for the transition between the inactive and active states. Several strategiesmay be used to adjust the energy landscape, including modulating the affinity of the input signal of the sensor module by mutating the binding site, and / or mutating the coiled-coil transmission module operatively linked to an effector module (non-limiting examples of HK, luciferase or DNA binding modules). Accordingly, introducing mutations in the different components of the biosensors produces biosensor variants having different functional properties, variations in their dose-response curves, implying changes in the basal level, trigger threshold, dynamic range, and maximum activity (Figure 3). It is understood that such strategies may be implemented to engineer non-limiting examples of tunable HK biosensors, tunable DNA binding biosensors and tunable luciferase biosensors.

[0107] In some embodiments, the trigger threshold of the tunable biosensor is decreased by introducing at least one mutation destabilizing the closed conformation to favor the transient open conformation which induces a change in the functional status of the tunable chimeric biosensor (Figure 3). A destabilizing mutation destabilizes the coiled-coil structure such as the substitution of a Leu placed in an a and / or d position of the heptad repeat with a Ser. This substitution was shown to destabilize the coiled-coil structure in the tunable biosensor BioRapDesk_d2 (substitution L258S, according to the BioRapDesk numeration). A destabilizing mutation may advantageously increase the binding affinity of the sensor module for its input signal, and / or favor interactions between the first sensor domain and the second sensor domain of the sensor module.

[0108] In tunable biosensors comprising one or more TD, destabilizing mutations may destabilize interactions between the transmembrane domains of the dimer. A non- limiting example of mutation includes S284A as exemplified in m5BioRapDesk (SEQ ID NO: 69).

[0109] One or more destabilizing mutation may also modulate the interactions between the DHp domain and the ABD domain. Non-limiting examples of mutation destabilizing DHp-ABD interactions in BioRapDesk include E31Q, L336A, R441Q, F444A or F444L.

[0110] In tunable HK biosensors where the functional status of the close conformation is characterized by an auto-kinase activity, a destabilizing mutation favors rearrangement to an open conformation, induces the inactivation of the auto-kinase, thereby resulting in the inactivation of the tunable HK biosensor. In one aspect of the embodiments, the functional status of the open conformation of the tunable HK biosensor is characterized by a phosphatase activity. Non-limiting mutations that weaken the coiled coil, includeM247A, M247S, M251A, M251S, L254A or L254S (numeration according to the different BioRapPhoq variants).

[0111] In tunable HK biosensors where the functional state of the closed conformation is characterized by a phosphatase activity, a destabilizing mutation favors rearrangement to an open conformation which induces the inactivation of the phosphatase. In one aspect of the embodiment, the functional status of the open conformation is characterized by an auto-kinase activity e.g., BioRapDesk_d2 (SEQ ID NO: 30).

[0112] In tunable HK biosensors where the functional state of the closed conformation is characterized by an inactive auto-kinase activity, a destabilizing mutation favors rearrangement to an open conformation which induces the activation of the auto- kinase, thereby resulting in the activation of the biosensor with lower levels of signal e.g., BioRapDesk_d2 (SEQ ID NO: 30).

[0113] In tunable luciferase biosensors, where the closed conformation of the dimer precludes the interaction between the split fragments of a luciferase enzyme, and the self- assembling into a functional enzyme, a destabilizing mutation favors rearrangement to an open conformation allowing the split fragments to assemble into functional enzyme capable of emitting luminescence in the presence of a substrate. In preferred embodiments, stabilizing mutations such as E254A, E254V or E254L (according to BioRapDHpLuc numeration) or destabilizing mutations, like L251A or L251S, are expected to generate changes in the energetic landscape, hindering or facilitating the switching mechanism from one state to the other, respectively.

[0114] In some embodiments, the trigger threshold of the tunable biosensor is increased by introducing at least one mutation stabilizing the closed conformation associated with a functional status characterized by an active or inactive function. A stabilizing mutation may advantageously stabilize the coiled-coil structure. A substitution of a Ser to Leu in a key position of a heptad repeat improves the coiled-coil heptad periodicity as exemplified in biosensor BioRapDesk_s1 (SEQ ID NO: 32). The addition of a second mutation Ala to Leu in the heptad repeat as exemplified in biosensor BioRapDesk_s2 improves hydrophobic packing (SEQ ID NO: 34). The insertion of an additional heptad repeat derived from GCN4 ideal coiled-coil motif (SEQ ID NO: 80) stabilizes closed conformation as exemplified in biosensor BioRapDesk_s3 (SEQ ID NO: 36).

[0115] In some aspects of the embodiments, a stabilizing mutation may be introduced to decrease the binding affinity of the sensor module for its input signal, and / or decrease the number of interactions between the first sensor domain and the second sensor domain of thesensor module. In tunable biosensor comprising TD such as a tunable periplasmic biosensor, a stabilizing mutation that promotes packing stabilizes transmembrane interactions.

[0116] In tunable HK biosensors, the functional status is characterized by an active or inactive auto-kinase and / or an active or inactive phosphatase.

[0117] In tunable HK biosensors, a stabilizing mutation may also be introduced to favor interactions between the DHp domain and the ADB domain. A non-limiting example includes the substitutions T288V (according to the BioRapDesk numeration).

[0118] Repurposing the tunable biosensors to different input signals

[0119] For periplasmic binding protein (PBP) as sensor module, a non-limiting example of PBP is the periplasmic binding protein of ArtJ from the thermophilic bacterium Geobacillus stearothermophilus . In one embodiment, the ArtJ periplasmic binding protein domain (PDB Id 2Q2A, left panel) was circularly permutated, where β4 was connected with β10 and β9 with β5, the loop α6p9 was cut and a 9-residue flexible linker between the N- and C-terminal lobs of the PBP module was inserted between α8 and β9 (Figure 4A). This modified PBP (Figure 4a; right panel) has the a6 helix at the C-terminus. It constitutes a sensor module against arginine and was named BioArg. The structural models are depicted as cartoon colored with a ramp from N-terminus (Figure 4A; in blue) to C-terminus (Figure 4A; in red). Arginine molecules are shown in stick representation (Figure 4A). BioArgDesk design was constructed similarly to BioRapDesk where the C-terminal domain of the sensor module was connected to DesKC through a rigid helical linker and stabilized in the phosphatase state (Figure 4B). Alphafold modeling suggested that our design should correctly fold as a phosphatase state in the absence of the ligand (Figure 4B). Structural model of the full biosensor in different states were generated through Alphafold2 in the phosphatase and phosphotransferase conformation, using 7SSJ and 5IUM crystal structures as templates and manual building (Figure 4B). Figure 4C shows the arginine dose-response curve of BioArgDesk. Purified BioArgDesk was incubated with ATP (1 mM), magnesium chloride (5 mM) and varying concentrations of arginine (0-10 mM), at pH 8, 25 °C, during 15 min (Figure 4C). The degree of phosphorylation was determined by Phostag SDS-PAGE gels and densitometry. A Hill equation was fitted to the data corresponding to three independent experiments (Figure 4C). . BioArgDesk was inactive in the absence of arginine, the cognate signal of ArtJ, and activated in its presence (Figure 4C).

[0120] In-silico methods for designing tunable biosensors

[0121] An alternative approach of natural ligand-binding domains as sensor module is by computationally design ligand-binding domains.

[0122] A method for designing tunable dimeric biosensors against macromolecules involves design the sensor module with motif scaffolding with RF diffusion (Watson et al. 2023) or fully denovo with RF diffusion (Watson et al. 2023), BindCraft (Pacesa et al. 2024), among others (steps 1-2). After filtering (step 3), the best binders are integrated at the N- terminus of the biosensor and the compatibility with the closed conformation of the biosensor is evaluated using protein modeling algorithms (step 4), like Alphafold. Finally, the selected biosensors are experimentally tested (step 5) (Figure 10). As a proof of concept, we validated the ability of biosensors to recognize proteins by generating ligands against FRB domain (SEQ ID NO 26) using BindCraft (Figure 11). Protein binders (Figure 11 A; shown as cartoon in grey) against FRB (Figure 11 A; depicted as cartoon in green) were denovo designed using BindCraft (Pacesaet al. 2024). The DNA of a set of eleven binders (SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111) were synthesized and inserted in bacterial expression plasmid compatible with the bacteria BioRapDesk biosensor system (Figure 11 A). E. coli cells were co-transformed with the plasmid including BioRapDesk biosensor and with the plasmids for the different designed binder proteins, diluted to 0.1 OD600and incubated in a plate reader (TEC AN 200 Pro) at 37 °C with agitation (Figure 11B). Fluorescence levels (λex= 475 nm, λem= 512 nm) (normalized by OD450) after 6 h of incubation were measured. Data correspond to triplicates of a representative experiment. At least four of the eleven designed protein binders triggered the kinase activity of the biosensor and expression of the reporter (Figure 11B).

[0123] There are three bioinformatic methods for designing tunable dimeric biosensors against small molecules, comprising a sensor module and an effector module, including generating a sensor module, the sensor module comprising two sensor domains, domain A and domain B, capable of assembling in a closed conformation to form a ligand binding site upon recognizing the ligand. Method 1 is disclosed in Figure 5 involves the denovo design of sensor modules. A ligand binding site is is defined (step 1), from a known structure or from a theoretically constructed structure to optimize residuesligand contacts (step 1) (Lucas & Kortemme, 2020). The ligand binding site is then divided in two halves, the first set of ligand binding residues and a second non-overlapping set of ligand binding residues (step 2). The first set of ligand binding residues and a helical segment are embeddedwithin a domain B, designed by partial diffusion or inpainting (Watson et al., 2023) and ProteinMPNN (Dauparas et al., 2022) or LigandMPNN (Dauparas et al., 2023) to allow the folding of the first set of ligand binding residues and the helical segment into a functional three-dimensional structure such as the helical segment is placed at the C-terminus of domain B, is positioned to favor the formation of a dimer in a closed conformation and the first set of the ligand binding residues is oriented towards the dimer interface upon dimerization (step 3 and 4). The second non-overlapping set of ligand binding residues is embedding in a new domain, designated as domain A, and designed by partial diffusion or inpainting and ProteinMPNN or LigandMPNN to allow the folding of the second non-overlapping set of ligand binding residues into a functional three-dimensional conformation structure such as the first set of ligand binding residues and the second non-overlapping set of ligand binding residues form a functional ligand binding site capable of accommodating the ligand, and domain A and domain B assemble into a functional sensor module capable of adopting an open or closed conformation (step 5). The module sensor is operably linked to the effector module through a helical linker comprising at least one heptad repeat (step 5). The final homodimeric structure is selected and validated experimentally. Methods 2 and 3 relate to alternative pipelines that were used in the invention to generate biosensors against small molecules (Figure 12). Method 2 is depicted in Figure 12A, where the first pipeline starts with the placing of different ligand conformers randomly oriented at the center, between domain A and B, of BioArg sensor module (step 1). The ligand binding site is then reengineered using different types of algorithms such as RFdiffusionAAT (Krishna et al. 2024) and LigandMPNN (Dauparas et al., 2023), PocketGen (Zhang et al. 2024) or Rosetta Suite (Leman et al. 2020) (step 2). The design of the pocket undergoes an iterative process and includes three stages: i) sequence design (step 3); ii) backbone and ligand complex refinement (step 4); and iii) decoy assessment and filtering (step 5). After at least two iterations, models are selected for experimental validation (step 6). Method 3 (Figure 12B) relates to the second pipeline that involves searching a ligand binding site (Zanghellini et al. 2006, Cao et al. 2022) over a scaffold library generated using partial RF diffusion (Watson et al. 2023) (step 1). The iterative ligand binding design process is followed (steps 2-4) and the selected models are experimentally validated (step 5).

[0124] Preferably, ligand binding site (Figure 5 and 12B) is identified using available structures and / or using a molecular docking algorithm and / or artificial intelligence algorithms.

[0125] As a proof of concept, functional biosensors against lactate were designed and validated using the in-silico methods described above (Figure 12A-D). Lactate biosensor design (BioLacDesk) was tested as proof of concept (Figure 12C). Different metrics from LigandMPNN (Dauparas et al. 2023) and Rosetta Suite were used to evaluate and filter decoys (Figure 12C). Screening of eight BioLacDesk biosensors designed were assessed where transformed E. coli cells with the different biosensor plasmids were diluted to 0.1 OD600and incubated in presence or absence of lactate in a plate reader (TEC AN 200 Pro) at 37 °C with agitation (Figure 12D). Fluorescence levels (λex= 475 nm, λem= 512 nm) (normalized by OD450) after 3.5 h of incubation in the absence (black) or presence (grey) of lactate (10 mM) were compared. Data correspond to triplicates of a representative experiment. Four of the eight biosensors (1, 2, 3 and 7) responded to lactate successfully (Figure 12D).

[0126] In an embodiment, an expression vector may be genetically engineered to incorporate the nucleic acid sequence encoding a protomer. Expression vectors may be selected from those readily available for use in prokaryotic or eukaryotic expression systems.

[0127] Two-component systems

[0128] HK participates in three different catalytic reactions, RR dephosphorylation, autophosphorylation and phosphotransfer, which are associated with three distinct structural states, phosphatase, auto-kinase and phosphotransferase, respectively (Buschiazzo & Trajtenberg, 2019). In the absence of stimulus, the HK promotes RR dephosphorylation (phosphatase activity), turning the pathway off. However, in the presence of its cognate signal, the HK shifts to the auto-kinase state, triggering the autophosphorylation of a conserved His. HK phosphorylation elicits a conformational change, adopting the phosphotransferase state and subsequently the phosphoryl-group is transferred to an aspartate of the cognate RR, stabilizing its dimerization and initiating the adaptative response.

[0129] In many transduction systems, additional components are included in the phosphorylation cascade, creating the so-called phosphorelay systems. The extra phosphoryl- transfer steps allow the creation of richer signaling networks, including extra signal modulators, such as dedicated phosphatases (Silversmith, 2010). Phosphorelays are usually involved in highly complex sensorial systems, like the Bacillus subtilis sporulation pathway.

[0130] Although great advance has been done in the Synthetic Biology field to construct complex circuits using well-known transcription factors, the repertoire of well characterized sensory proteins is still limited. The design of proteins with desired ligand-binding capacity is still a difficult challenge. TCSs are natural sensor and information processing systems that could become relevant tools in Synthetic Biology (Nieves et al., 2023). The implementation of TCS circuits in bacteria (Daeffler et al., 2017), yeast (Mishra et al. 2021) and mammalian cells (Maze & Benenson 2020, Scheller et al. 2020) has already been shown. A relevant property of TCS is that many HKs are transmembrane proteins, with the sensor domain facing the periplasmic space. Thus, signals are detected directly from the extracellular space, without the need for a specific transport mechanism. The modular architecture of the TCS proteins (Buschiazzo & Trajtenberg, 2019) are also ideal for protein engineering campaigns. Lastly, the versatility of the phosphoryl-transfer chemistry enables different configuration of its reversibility, adding another layer of complexity in the signal transmission (Lima et al. 2023). We can now recognize that engineering simple two- component pathways all the way to highly complex phosphorelays is becoming feasible.

[0131] In some embodiments, a two-component system (TCS) includes: a tunable biosensor, and a cognate response regulator (RR) that includes a receiver domain, where the activated biosensor transfers a phosphoryl moiety to a phosphorylation site of the cognate RR. Frequently, the receiver domain of RR is coupled with a DNA binding domain as an effector module.

[0132] A TCS of the invention may be introduced into a prokaryotic or eukaryotic cell to modulate the expression of a target gene, or the catalytic activity of a target protein. In such embodiments, the RR of the TCS, once phosphorylated, dimerize and bind to the promoter of the target gene, thereby affecting its transcription.

[0133] A biocomputing system can be created in a prokaryotic and eukaryotic cell. Such biocomputing system is made of a plurality of tunable biosensors associated with its cognate RR (Figure 15A-D). Starting from our experimentally parametrized kinetic models of the DesK / DesR system (Lima et al. 2023) we constructed different kinetic models of circuits and run simulations to characterize their behavior under different concentrations of signal inputs. Simulations of a system including two different HK based biosensors that recognize the same RR and are activated by distinct signals (s1 and s2). The system is connected through phosphorylation and / or dephosphorylation reactions in order to integrate signals coming from different biosensors. The connectivity between different components can be engineered to perform different type of logic computations that will be associated to a set of programmed responses. Accordingly, the engineered network can trigger genetic or biochemical programs based on the incoming information. For example, the system behaves as AND gate showing accumulation of phosphorylated RR after 10 min of simulations, andthus activated, only in the presence of higher concentrations of signals s1 and s2 (Figure 15 A), and when using biosensors mutated to minimize the phosphatase activity, is turned on either in the presence of s1 or s2 (OR gate) (Figure 15B), and when simulations are similar to circuits as in igure 15A and Figure 15B but include module that inverts the activation of the system in the presence of the signal, NAND and NOR gates are generated (Figures 15C-D).

[0134] Procaryotic cells or eukaryotic cells that comprised a TCS or a biocomputing system may be developed for applications ranging from diagnostics, therapeutic, bioproduction, agriculture, bioremediation or climate crisis.EXAMPLES

[0135] Specific embodiments will now be demonstrated by reference to the following examples. It should be understood that these examples are disclosed solely by way of illustrating the invention and should not be taken in any way to limit the scope of the present invention.

[0136] Materials and Methods - Protein design

[0137] The BioRapDesk (SEQ ID NO: 29) was constructed by grafting a FKBP51- FRB construct as a sensor domain (SEQ ID NO: 27) at the N-terminus of DesKC (SEQ ID NO: 28) (cytoplasmic and catalytically active region). FKBP51-FRB construct (Figure 1E- H) involves the FKBP51 domain linked to FRB through a 13-residue flexible linker (GSSASGTSSTSSG: SEQ ID NO: 81). The grafting sampling was performed by testing different FRB models, with variable α-helical extension at the C-terminus, in silico attached to DesKC at different positions. This allowed for testing several possible orientations of the sensor region. Solutions that place the rapamycin binding site facing the dimerization interface were tested using Alphafold (Jumper et al., 2021). The best solution includes a 6 residues α-helical linker before the KSRKERER motif in DesK (residue 152). To promote the phosphatase state in the absence of rapamycin, the sequence of the linker was designed to extend the N-terminal coiled-coil heptad repeat of DesKC.

[0138] Building biosensors according to the in-silico method

[0139] Biosensors recognizing any small molecule ligand can be generated following Methods 1 (Figure 5), 2 or 3 (Figure 12A and B). Method 1 involves following a few steps: i) build an ideal binding site; ii) split the binding site in two halves, ensuring enough contacts in both hemi-sites; iii) for one of the half-binding sites, we added an α-helix, oriented in a way that when placed in the biosensor, the binding site will be at the dimerization interface; iv)create a scaffold for the first hemi-binding site using RFdiffusion (Watson et al., 2023) and ProteinMPNN (Dauparas et al., 2022) in the context of a symmetric dimer, fixing the position and the sequence of binding site motif; and v) construct the scaffold protein of the second half-binding site in the context of the de novo domain constructed in step iv. Methods 2 and 3 use ligand binding proteins as template, non-limiting examples is includes BioRap (SEQ ID NO 27), BioArg (SEQ ID NO 45) or BioAba (SEQ ID NO 49) sensor modules, to reengineer the ligand binding site using an iterative protein design procedures depicted in Figure 12A-B.

[0140] Biosensors recognizing any protein ligand can be generated following the procedure shown in Figure 10, following a few steps: the sensor module to a target molecule is designed using an iterative process using RFdiffusion (Watson et al. 2023), ProteinMPNN (Dauparas et al., 2022), and Alphafold (Jumper et al., 2021), or BindCraft (Pacesa et al. 2024), among others. Denovo binders are then tested for implementation as sensor modules in the biosensor through modeling with Alphafold (Jumper et al., 2021).

[0141] Strains and plasmids

[0142] All recombinant proteins were cloned in pCOLA-Duet plasmid including a N- terminally His6-tagged fusions. For in vivo functional evaluation we generated plasmids also using pCOLA-Duet1 as backbone. For example, pCOLA-BioRapDesK includes GFP under Pdes promoter and an operon coding for BioRapDesk followed by DesR under the expression of a T51ac promoter. All DNA fragments were synthesized and subcloned at Genscript. Recombinant protein expression and biosensor functional characterization in vivo was performed in Escherichia coli strain TOP10F’ (Figure 9).

[0143] The same or similar constructs can be included in different type of plasmids or inserted in the genome of the cells.

[0144] Autophosphorylation and phosphatase assays

[0145] Recombinant protein purification was performed using well-established protocols (Lima et al. 2023). In brief, proteins were purified using Ni-NTA chromatography, followed by TEV cleavage. Subsequently, samples were loaded in a size exclusion chromatography (SEC) using a HiLoad 16 / 60 Superdex200 preparation grade column (GE Healthcare) equilibrated with a buffer consisting of 20 mM tris-HCl (pH 8.0) and 0.3 M NaCl (SEC buffer). The resulting proteins were concentrated to approximately 10 mg / ml and stored at -80 °C. Biosensor autophosphorylation activity was measured by using a coupled enzyme assay as performed previously (Trajtenberg et al. 2010), or the degree of phosphorylation was measured through Phostag SDS-PAGE (Lima et al. 2023).

[0146] In vivo biosensor characterization (reporter assay)

[0147] Biosensor characterization in vivo was performed by transforming TOP10F’ or BW27558 (AphoQ-phoP obtained from the E. coli Genetic Resource Center) cells with the corresponding plasmids.Exponential growing cells in M9 media were plate in 96well plates and incubated with different concentrations of ligands (like rapamycin). The fluorescence was followed in a plate reader (Varioskan or Tecan) with agitation and at 37 °C.

[0148] Biosensor constructsTunable Membrane BiosensorsBioRapDHpLuc or BioRapDHpFAST: DHp domain may help to dimerize and function as a switch.

[0149] Exemplary embodiments of the tunable biosensorsI. Biosensor BioRapDesk

[0150] Biosensor BioRapDesk (SEQ ID NO: 29) and its variants comprise a BioRap sensor module (SEQ ID NO: 27) (sensor domains FKBP51 (SEQ ID NO: 25) and FRB (SEQ ID NO: 26) fused through a flexible linker (e.g., SEQ ID NO: 81 or SEQ ID NO: 20) operably linked to a Histidine Kinase (HK) module derived from Desk (SEQ ID NO: 28)). In some embodiments, tunable HK biosensor BioRapDesk variants are constructed by introducing mutations at one or more critical positions 258, 251, 265 of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor, e.g., BioRapDesk _d1, _d2, _s1 variants comprising the sequence SEQ ID NO: 41, SEQ ID NO: 30, SEQ ID NO: 32, respectively. In some embodiments, the sensor module BioRap and the HK module comprising a sequence selected from the group consisting of DesKC (SEQ ID NO: 28), Desk_d2 (SEQ ID NO: 31), Desk_s1 (SEQ ID NO: 33), or Desk_d1 (SEQ ID NO: 42) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.Sensor domain FKBP51 (SEQ ID NO: 25)Sensor domain FRB (SEQ ID NO: 26)Sensor module BioRap (SEQ ID NO: 27)HK Module DesKC (SEQ ID NO: 28)BioRapDesk (SEQ ID NO: 29)BioRapDesk_d2 (SEQ ID NO: 30) (Destabilizing Substitution L258S)Desk_d2 (SEQ ID NO: 31) (Destabilizing Substitution L258S)BioRapDesk_s1 (SEQ ID NO: 32) (Stabilizing substitution S251L)Desk_s1 (SEQ ID NO: 33) (Stabilizing substitution S251L)BioRapDesk_s2 (SEQ ID NO: 34) (Stabilizing substitutions S251L / A265L)Desk_s2 (SEQ ID NO: 35) (Stabilizing substitutions S251L / A265L)BioRapDesk_s3 (SEQ ID NO: 36)BioRapDesk_s4 (Stabilizing substitution S251A) (SEQ ID NO: 37)Desk_s4 (SEQ ID NO: 38) (Stabilizing substitution S251A)BioRapDesk_s5 (Stabilizing substitution S251V) (SEQ ID NO: 39)Desk_s5 (SEQ ID NO: 40) (Stabilizing substitution S251V)BioRapDesk_d1 (Destabilizing substitution A265V) (SEQ ID NO: 41) Desk_d1 (SEQ ID NO: 42) (Destabilizing substitution A265V)II. Biosensor BioArgDesk

[0151] Biosensor BioArgDesk (SEQ ID NO: 46) comprises a BioArg sensor module (SEQ ID NO: 45) derived from the substrate binding component of ArtJ, a periplasmic binding protein, operably linked to a HK module derived from Desk (DeskC, SEQ ID NO: 28). In some embodiments, tunable HK biosensor BioArgDesk variants are constructed by introducing mutations at one or more critical positions 258, 251, 265 of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In some embodiments, the sensor module BioArg (SEQ ID NO: 45) and the HK module comprising a sequence selected from the group consisting of DesKC (SEQ ID NO: 28), Desk_d2 (SEQ ID NO: 31), Desk_s1 (SEQ ID NO: 33) or Desk_d1 (SEQ ID NO: 42) are operably linked through a helical linker comprising a sequence selected fromthe group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. The sensor module comprises the 2 lobes (SEQ ID NO: 43 and SEQ ID NO: 44) forming ArtJ substrate binding site which are connected to one another through a flexible linker comprising the sequence SEQ ID NO: 81 or SEQ ID NO: 20.Sensor domain derived from one of the 2 lobes / portions of the substrate binding component of ArtJ, a periplasmic binding protein (SEQ ID NO: 43) Sensor domain derived from the other lobe / portion of the substrate binding component of ArtJ, (SEQ ID NO:44) Sensor module BioArg (SEQ ID NO: 45) BioArgDesk (SEQ ID NO: 46)III. Biosensor BioAbaDesk

[0152] Biosensor BioAbaDesk (SEQ ID NO: 50) comprises a BioAba sensor module (SEQ ID NO: 49) based on PYL2 (SEQ ID NO: 47) and HAB1 (SEQ ID NO: 48), operably linked to a HK module (DesKC) derived from DesK (SEQ ID NO: 28). In some embodiments, tunable HK biosensor BioAbaDesk variants are constructed by introducing mutations at one or more critical positions 258, 251, 265 of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In some embodiments, the sensor module BioAba (SEQ ID NO: 49) and the HK module comprising a sequence selected from the group consisting of DesKC (SEQ ID NO: 28), Desk_d2 (SEQ ID NO: 31), Desk_s1 (SEQ ID NO: 33), Desk_s2 (SEQ ID NO: 35), Desk_s4 (SEQ ID NO: 38), Desk_s5 (SEQ D NO: 40), or Desk_d1 (SEQ ID NO: 42) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. PYL2 (SEQ ID NO: 47) and HAB1 (SEQ ID NO: 48) are connected to one another through a flexible linker comprising one of the sequence SEQ ID NO: 81 or SEQ ID NO: 20.Sensor domain derived from PYL2 (SEQ ID NO: 47)Sensor domain derived from HAB1 (SEQ ID NO: 48)Sensor module BioAba (SEQ ID NO: 49) BioAbaDesk (SEQ ID NO: 50)IV. Biosensors BioLacDesk

[0153] Biosensors BioLacDesk (SEQ ID NO: 88, SEQ ID NO: 89, SEQ IDNO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95) comprises a BioLac sensor module (SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134) designed from a PBP protein, operably linked to a HK module (DesKC) derived from DesK (SEQ ID NO: 28). In some embodiments, tunable HK biosensors BioLacDesk variants are constructed by introducing mutations at one or more critical positions 258, 251, 265 of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In some embodiments, the sensor module BioLac and the HK module comprising a sequence selected from the group consisting of DesKC (SEQ ID NO: 28), Desk_d2 (SEQ ID NO: 31), Desk_s1 (SEQ ID NO: 33), Desk_s2 (SEQ ID NO: 35), Desk_s4 (SEQ ID NO: 38), Desk_s5 (SEQ D NO: 40), or Desk_d1 (SEQ ID NO: 42) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19.Sensor module BioLac1 (SEQ ID NO: 127) Sensor module BioLac2 (SEQ ID NO: 128) Sensor module BioLac3 (SEQ ID NO: 129) Sensor module BioLac4 (SEQ ID NO: 130) Sensor module BioLac5 (SEQ ID NO: 131) Sensor module BioLac6 (SEQ ID NO: 132) Sensor module BioLac7 (SEQ ID NO: 133) Sensor module BioLac8 (SEQ ID NO: 134) BioLaclDesk (SEQ ID NO: 88) BioLac2Desk (SEQ ID NO: 89) BioLac3Desk (SEQ ID NO: 90) BioLac4Desk (SEQ ID NO: 91) BioLac5Desk (SEQ ID NO: 92)BioLac6Desk (SEQ ID NO: 93)BioLac7Desk (SEQ ID NO: 94)BioLac8Desk (SEQ ID NO: 95)V.. Biosensor BioRapNarx

[0154] Biosensor BioRapNarx (SEQ ID NO: 52) comprises a BioRap sensor module (sensor domains FKBP51 / FRB linked through a flexible linker) operably linked to a HK module derived from HisKA_3 (NarXC, SEQ ID NO: 51). In some embodiments, tunable HK biosensor BioRapNarx variants are constructed by introducing mutations at one or more critical positions of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In other embodiments, the sensor module BioRap (SEQ ID NO: 27) and the NarXC effector module (comprising SEQ ID NO: 51) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the sensor module BioRap may be substituted with the sensor module BioArg (SEQ ID NO: 45), BioAba (SEQ ID NO: 49), BioLacl (SEQ ID NO: 127), BioLac2 (SEQ ID NO: 128), BioLac3 (SEQ ID NO: 129), BioLac4 (SEQ ID NO: 130), BioLac5 (SEQ ID NO: 131), BioLac6 (SEQ ID NO: 132), BioLac7 (SEQ ID NO: 133) or BioLac8 (SEQ ID NO: 134) to construct biosensor BioRapNarx, biosensor BioAbaNarx, biosensor BioLaclNarx, biosensor BioLac2Narx, biosensor BioLac3Narx, biosensor BioLac4Narx, biosensor BioLac5Narx, biosensor BioLac6Narx, biosensor BioLac7Narx or biosensor BioLac8Narx, respectively.HK module NarXC (SEQ ID NO: 51) BioRapNarx (SEQ ID NO: 52)VI, Biosensors BioRapKinal / BioRapKinal

[0155] Biosensors BioRapKinal (SEQ ID NO: 54) and BioRapKina2 (SEQID NO: 55) comprise a BioRap sensor module (SEQ ID NO: 27) (sensor domains FKBP51 / FRB linked through a flexible linker of SEQ ID NO: 81 or SEQ ID NO: 20) operably linked to a HK module derived from HisKA (KinAC1 and KinAC2). In some embodiments, tunable HK biosensor BioRapNarX variants are constructed by introducingmutations at one or more critical positions of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In other embodiments, the sensor module BioRap (SEQ ID NO: 27) and the HK effector module (comprising SEQ ID NO: 53) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the sensor module BioRap may be substituted with the sensor module BioArg (SEQ ID NO: 45), BioAba (SEQ ID NO: 49), BioLacl (SEQ ID NO: 127), BioLac2 (SEQ ID NO: 128), BioLac3 (SEQ ID NO: 129), BioLac4 (SEQ ID NO: 130), BioLac5 (SEQ ID NO: 131), BioLac6 (SEQ ID NO: 132), BioLac7 (SEQ ID NO: 133) or BioLac8 (SEQ ID NO: 134) to construct biosensors BioArgKinal, BioArgKina2, BioAbaKinal, BioAbaKina2, BioLaclKinal, BioLaclKina2, BioLac2Kinal, BioLac2Kina2, BioLac3Kinal, BioLac3Kina2, BioLac4Kinal, BioLac4Kina2, BioLac5Kinal, BioLac5Kina2, BioLac6Kinal, BioLac6Kina2, BioLac7Kinal, BioLac7Kina2, BioLac8Kinal or BioLac8Kina2.,Effector module of HisKA, KinAC (SEQ ID NO: 53)BioRapKinal (SEQ ID NO: 54) BioRapKina2 (SEQ ID NO: 55)VII. Biosensors BioRapPhoq

[0156] Biosensors BioRapPhoq comprises a BioRap sensor module (SEQ IDNO:27) (sensor domains FKBP51 / FRB linked through a flexible linker) operably linked to a HK module PhoQC1 (SEQ ID NO: 57) or PhoQC2 (SEQ ID NO: 59). In some embodiments, tunable HK biosensor BioRapPhoq variants are constructed by introducing mutations at one or more critical positions of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In other embodiments, the sensor module BioRap (SEQ ID NO: 27) and the HK effector module (comprising SEQ ID NO: 57 or SEQ ID NO: 59) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 123. In some embodiments, the sensor module BioRap may be substituted with either of the sensor modules BioArg(SEQ ID NO: 45), BioAba (SEQ ID NO: 49), BioLacl (SEQ ID NO: 127), BioLac2 (SEQ ID NO: 128), BioLac3 (SEQ ID NO: 129), BioLac4 (SEQ ID NO: 130), BioLac5 (SEQ ID NO: 131), BioLac6 (SEQ ID NO: 132), BioLac7 (SEQ ID NO: 133) or BioLac8 (SEQ ID NO: 134) to construct biosensors BioArgPhoq, BioAbaPhoq, BioLac1Phoq, BioLac2Phoq, BioLac3Phoq, BioLac4Phoq, BioLac5Phoq, BioLac6Phoq, BioLac7Phoq or BioLac8Phoq, using either PhoQC1 (SEQ ID NO: 57) or PhoQC2 (SEQ ID NO: 59).PhoQC1 (SEQ ID NO: 57) PhoQC2 (SEQ ID NO: 59) BioRapPhoq (SEQ ID NO: 56) BioRapPhoq1 (SEQ ID NO: 97) BioRapPhoq2 (SEQ ID NO: 58) BioRapPhoq3 (SEQ ID NO: 60) BioRapPhoq4 (SEQ ID NO: 61) BioRapPhoq5 (SEQ ID NO: 62) BioRapPhoq6 (SEQ ID NO: 63) BioRapPhoq7 (SEQ ID NO: 64)III. Biosensor BioRapFixI

[0157] Biosensor BioRapFixI comprises a BioRap sensor module (SEQ IDNO:27) (sensor domains FKBP51 / FRB linked through a flexible linker) operably linked to a HK module FixLC (SEQ ID NO: 117). In some embodiments, tunable HK biosensor BioRapFixI variants are constructed by introducing mutations at one or more critical positions of the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. In other embodiments, the sensor module BioRap (SEQ ID NO: 27) and the HK effector module (comprising SEQ ID NO: 57 or SEQ ID NO: 59) are operably linked through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 124. In some embodiments, the sensor module BioRap may be substituted with either of the sensor modules BioArg (SEQ ID NO: 45), BioAba (SEQ ID NO: 49), BioLacl (SEQ ID NO: 127), BioLac2 (SEQ ID NO: 128), BioLac3 (SEQ ID NO: 129), BioLac4 (SEQ ID NO: 130), BioLac5 (SEQ ID NO: 131), BioLac6 (SEQ IDNO: 132), BioLac7 (SEQ ID NO: 133) or BioLac8 (SEQ ID NO: 134) to construct biosensors BioArgFix1, BioAbaFix1, BioLaclFix1, BioLac2Fix1, BioLac3Fix1, BioLac4Fix1, BioLac5Fix1, BioLac6Fix1, BioLac7Fix1 or BioLac8Fix1, respectively.FixLC (SEQ ID NO: 117)BioRapFix1 (SEQ ID NO: 87) 1X»

[0158] Membrane biosensors comprise at least one transmembrane domainTD comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 84, SEQ ID NO: 85. In some embodiments, the membrane biosensors may further comprise a signal peptide comprising a sequence selecting from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 86, SEQ ID NO: 120. Biosensors may be constructed using any of the sensor module BioRap (SEQ ID NO: 27), BioArg (SEQ ID NO: 45), BioAba (SEQ ID NO: 49), BioLacl (SEQ ID NO: 127), BioLac2 (SEQ ID NO: 128), BioLac3 (SEQ ID NO: 129), BioLac4 (SEQ ID NO: 130), BioLac5 (SEQ ID NO: 131), BioLac6 (SEQ ID NO: 132), BioLac7 (SEQ ID NO: 133) or BioLac8 (SEQ ID NO: 134) operably linked to any of the HK effector DesKC (SEQ ID NO: 28), NarXC (SEQ ID NO: 51), PhoQC1 (SEQ ID NO: 57), PhoQC2 (SEQ ID NO: 59), KinAC (SEQ ID NO: 53), FixLC (SEQ ID NO: 117) or any of their variants Desk_d2 (SEQ ID NO: 31), Desk_s1 (SEQ ID NO: 33), Desk_s2 (SEQ ID NO: 35), Desk_s4 (SEQ ID NO: 38), Desk_s5 (SEQ D NO: 40), or Desk_d1 (SEQ ID NO: 42) through a helical linker comprising a sequence selected from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 125 or SEQ ID NO: 126.

[0159] In some embodiments, tunable membrane biosensor variants are constructed by introducing mutations at one or more critical positions in the HK module and / or in the helical linker to modulate the trigger threshold of the tunable biosensor. m1BioRapDesk (SEQ ID NO: 65) m2BioRapDesk (NarX2-BioRap-NarX1_Desk) (SEQ ID NO: 66) m3BioRapDesk (DsbA-BioRap-NarX1_Desk) (SEQ ID NO: 67) m4BioRapDesk (CyoA-BioRap-NarX1_Desk) (SEQ ID NO: 68)m5BioRapDesk (DsbA-BioRap-CadC Desk) (SEQ ID NO: 69) m6BioRapDesk (CyoA-BioRap-CadC Desk) (SEQ ID NO: 70) m7BioRapDesk (DsbA-BioRap-mlL22_Desk) (SEQ ID NO: 71) m8BioRapDesk (CyoA-BioRap-mlL22_Desk) (SEQ ID NO: 72) m9BioRapDesk (BT4663-BioRap-BT4663_Desk) (SEQ ID NO: 98) m10BioArgDesk (BT4663-BioArg-L26_Desk) (SEQ ID NO: 112) m11BioRapDesk (BT4663-BioRap-GpA_Desk) (SEQ ID NO: 113)X. DNA binding domain (DBD) biosensors

[0160] Biosensors may be constructed using any of the sensor modules BioRap (SEQ ID NO: 27), BioArg (SEQ ID NO: 45) or BioAba (SEQ ID NO: 49) operably linked to the DBD effector (SEQ ID NO: 73) with an helical linker comprising a sequence selecting from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, DBD biosensor variants are constructed by introducing mutations at one or more critical positions in the DBD module and / or in the helical linker to modulate the trigger threshold of the tunable DBD biosensor.DBD (SEQ ID NO: 73) BioRapDBD (SEQ ID NO: 74)XI. Split biosensors

[0161] Luciferase Biosensors may be constructed using any of the sensor modules BioRap (SEQ ID NO: 27), BioArg (SEQ ID NO: 45) or BioAba (SEQ ID NO: 49) operably linked to the Luciferase effector with an helical linker comprising a sequence selecting from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 99. The luciferase effector may comprise two fragments SmBit (comprising SEQ ID NO: 75) and LgBit (comprising SEQ ID NO: 76) linked through at least a flexible linker of SEQ ID NO: 81 or SEQ ID NO: 20 and / or a helical linker as defined above. In some aspects of the embodiments, the biosensors further comprise a DHp domain (comprising SEQ ID NO: 77).

[0162] In some embodiments, luciferase biosensor variants are constructed by introducing mutations at one or more critical positions in the SmBit / LgBit module and / or in the helical linkers and / or the DHp domain, if present, to modulate the trigger threshold of the tunable biosensor. Exemplary luciferase biosensors are BioRapLuc (SEQ ID NO: 78) and BioRapDHpLuc (SEQ ID NO: 79).

[0163] FAST Biosensors may be constructed using any of the sensor modules BioRap (SEQ ID NO: 27), BioArg (SEQ ID NO: 45) or BioAba (SEQ ID NO: 49) operably linked to the FAST effector with an helical linker comprising a sequence selecting from the group consisting of amino acid sequence LLE, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 99. The FAST effector may comprise two fragments CFAST11 (comprising SEQ ID NO: 126) and NFAST (comprising SEQ ID NO: 116) linked through at least a flexible linker of SEQ ID NO: 81 or SEQ ID NO: 20 and / or a helical linker as defined above. In some aspects of the embodiments, the biosensors further comprise a DHp domain (comprising SEQ ID NO: 77).SmBit (SEQ ID NO: 75) LgBit (SEQ ID NO: 76) Histidine-phosphotransfer domain (DHp) domain (SEQ ID NO: 77) BioRapLuc (SEQ ID NO: 78) BioRapDHpLuc (SEQ ID NO: 79) CFAST11 (SEQ ID NO: 126) NFAST (SEQ ID NO: 116) BioRapDHpFAST (SEQ ID NO: 96)SCIENTIFIC LITTERATUREAlbanesi, D., Martin, M., Trajtenberg, F., Mansilla, M.C., Haouz, A., Alzari, P.M., de Mendoza, D., and Buschiazzo, A. (2009) Structural plasticity and catalysis regulation of a thermosensor histidine kinase. Proc Natl Acad Sci USA 106: 16185-16190.Ausmees, N., and Jacobs-Wagner, C. (2003) Spatial and temporal control of differentiation and cell cycle progression in Caulobacter crescentus. Annu Rev Microbiol 57: 225- 247.Banaszynski, L.A., Liu, C.W., and Wandless, T.J. (2005) Characterization of the FKBP.rapamycin.FRB ternary complex. Journal of the American Chemical Society 127: 4715-4721.Buschiazzo, A., and Trajtenberg, F. (2019) Two-Component Sensing and Regulation: How Do Histidine Kinases Talk with Response Regulators at the Molecular Level? Annu Rev Microbiol 73: 22.21-22.22.Cao, L., B. Coventry, I. Goreshnik, B. Huang, W. Sheffler, J. S. Park, K. M. Jude, I. Markovic, R. U. Kadam, K. H. G. Verschueren, K. Verstraete, S. T. R. Walsh, N. Bennett, A. Phal, A. Yang, L. Kozodoy, M. DeWitt, L. Picton, L. Miller, E. M. Strauch, N. D. DeBouver, A. Pires, A. K. Bera, S. Halabiya, B. Hammerson, W. Yang, S. Bernard, L. Stewart, I. A. Wilson, H. Ruohola-Baker, J. Schlessinger, S. Lee, S. N. Savvides, K. C. Garcia and D. Baker (2022). "Design of protein-binding proteins from the target structure alone." Nature 605(7910): 551-560.Daeffler, K.N., Galley, J.D., Sheth, R.U., Ortiz-Velez, L.C., Bibb, C.O., Shroyer, N.F., Britton, R.A., and Tabor, J. J. (2017) Engineering bacterial thiosulfate and tetrathionate sensors for detecting gut inflammation. Mol Sy st Biol 13: 923.Dauparas, J., Anishchenko, I., Bennett, N., Bai, H, Ragotte, R.J., Milles, L.F., Wicky, B.I.M., Courbet, A., de Haas, R.J., Bethel, N., Leung, P.J.Y., Huddy, T.F., Pellock, S., Tischer, D., Chan, F., Koepnick, B., Nguyen, H., Kang, A., Sankaran, B., Bera, A.K., King, N.P., and Baker, D. (2022) Robust deep learning-based protein sequence design using ProteinMPNN. Science 378: 49-56.Dauparas, J., G. R. Lee, R. Pecoraro, L. An, I. Anishchenko, C. Glasscock and D. Baker (2023). "Atomic context-conditioned protein sequence design using LigandMPNN." bioRxiv: 2023.2012.2022.573103.Dupre, E., B. Clantin, Y. Yuan, S. Lecher, E. Lesne, R. Antoine, V. Villeret and F. Jacob- Dubuisson (2021). "Structural insight into the role of the PAS domainfor signal transduction in sensor-kinase BvgS." J Bacteriol 203(9).Dixon, A.S., Schwinn, M.K., Hall, M.P., Zimmerman, K., Otto, P., Lubben, T.H., Butler, B.L., Binkowski, B.F., Machleidt, T., Kirkland, T.A., Wood, M.G., Eggers, C.T., Encell, L.P., and Wood, K.V. (2016) NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11: 400- 408.Gao, R., Bouillet, S., and Stock, A.M. (2019) Structural Basis of Response Regulator Function. Annu Rev Microbiol 73: 175-197.Gao, R., and Stock, A.M. (2017) Quantitative Kinetic Analyses of Shutting Off a Two- Component System. mBio 8.Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., Tunyasuvunakool, K., Bates, R., Zidek, A., and Potapenko, A. (2021) Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589.Kreil, T., Lacal, J., Busch, A., Silva-Jimenez, H., Guazzaroni, M.E., and Ramos, J.L. (2010) Bacterial sensor kinases: diversity in the recognition of environmental signals. Annu Rev Microbiol 64: 539-559.Krishna, R., J. Wang, W. Ahern, P. Sturmfels, P. Venkatesh, I. Kalvet, G. R. Lee, F. S. Morey-Burrows, I. Anishchenko, I. R. Humphreys, R. McHugh, D. Vafeados, X. Li, G. A. Sutherland, A. Hitchcock, C. N. Hunter, A. Kang, E. Brackenbrough, A. K. Bera, M. Baek, F. DiMaio and D. Baker (2024). "Generalized biomolecular modeling and design with RoseTTAFold All-Atom." Science 384(6693): eadl2528.Leaver-Fay, A., Jacak, R., Stranges, P.B., and Kuhlman, B. (2011) A generic program for multistate protein design. PloS one 6: e20937.Leman, J. K., B. D. Weitzner, S. M. Lewis, J. Adolf-Bryfogle, N. Alam, R. F. Alford, M. Aprahamian, D. Baker, K. A. Barlow, P. Barth, B. Basanta, B. J. Bender, K. Blacklock, J. Bonet, S. E. Boyken, P. Bradley, C. Bystroff, P. Conway, S. Cooper, B. E. Correia, B. Coventry, R. Das, R. M. De Jong, F. DiMaio, L. Dsilva, R. Dunbrack, A. S. Ford, B. Frenz, D. Y. Fu, C. Geniesse, L. Goldschmidt, R. Gowthaman, J. J. Gray, D. Gront, S. Guffy, S. Horowitz, P.-S. Huang, T. Huber, T. M. Jacobs, J. R. Jeliazkov, D. K. Johnson, K. Kappel, J. Karanicolas, H. Khakzad, K. R. Khar, S. D. Khare, F. Khatib, A. Khramushin, I. C. King, R. Kleffner, B. Koepnick, T. Kortemme, G. Kuenze, B. Kuhlman, D. Kuroda, J. W. Labonte, J. K. Lai, G. Lapidoth, A. Leaver-Fay, S. Lindert, T. Linsky, N. London, J. H. Lubin, S. Lyskov, J. Maguire, L. Malmstrbm, E. Marcos, O. Marcu, N. A. Marze, J. Meiler, R. Moretti, V. K. Mulligan, S. Nerli, C. Norn, S. 6’Conchuir, N. Ollikainen, S. Ovchinnikov, M. S. Pacella, X. Pan, H. Park, R. E. Pavlovicz, M. Pethe, B. G. Pierce, K. B. Pilla, B. Raveh, P. D. Renfrew, S. S. R. Burman, A. Rubenstein, M. F. Sauer, A. Scheck, W. Schief, O. Schueler-Furman, Y. Sedan, A. M. Sevy, N. G. Sgourakis, L. Shi, J. B. Siegel, D.-A. Silva, S. Smith, Y. Song, A. Stein, M. Szegedy, F. D. Teets, S. B. Thyme, R. Y.-R. Wang, A. Watkins, L. Zimmerman and R. Bonneau (2020). "Macromolecular modeling and design in Rosetta: recent methods and frameworks." Nature Methods 17(7): 665-680.Lima, S., Blanco, J., Olivieri, F., Imelio, J. A., Carrion, F., Alvarez, B., Buschiazzo, A., Marti, M., and Trajtenberg, F. (2023) Molecular basis of unidirectional information transmission in two-component systems: lessons from the DesK-DesR thermosensor. Set. Signal , 16,eabo7588 .Lubkowicz, D., Ho, C.L., Hwang, I.Y., Yew, W.S., Lee, Y.S., and Chang, M.W. (2018) Reprogramming Probiotic Lactobacillus reuteri as a Biosensor for Staphylococcus aureus Derived AIP-I Detection. ACS synthetic biology 7: 1229-1237.Lucas, J.E., and Kortemme, T. (2020) New computational protein design methods for de novo small molecule binding sites. PLoS computational biology 16: el008178.Maze, A. and Y. Benenson (2020). "Artificial signaling in mammalian cells enabled by prokaryotic two-component system." Nature Chemical Biology 16(2): 179-187.McClune, C.J., Alvarez-Buylla, A., Voigt, C.A., and Laub, M.T. (2019) Engineering orthogonal signalling pathways reveals the sparse occupancy of sequence space. Nature 574: 702-706.Mishra, D., Bepler, T., Teague, B., Berger, B., Broach, J., and Weiss, R. (2021) An engineered protein-phosphorylation toggle network with implications for endogenous network discovery. Science 373.Nieves, M., Buschiazzo, A., and Trajtenberg, F. (2023) Structural features of sensory two component systems: a synthetic biology perspective. Biochem J 480: 127-140.Ninfa, A.J., Selinsky, S., Perry, N., Atkins, S., Song, Q.X., Mayo, A., Arps, D., Woolf, P., and Atkinson, M.R., (2007) Using Two-Component Systems and Other Bacterial Regulatory Factors for the Fabrication of Synthetic Genetic Devices. In: Methods in enzymology. Elsevier, pp. 488-512.Pacesa, M., L. Nickel, C. Schellhaas, J. Schmidt, E. Pyatova, L. Kissling, P. Barendse, J. Choudhury, S. Kapoor, A. Alcaraz- Serna, Y. Cho, K. H. Ghamary, L. Vinue, B. J. Yachnin, A. M. Wollacott, S. Buckley, A. H. Westphal, S. Lindhoud, S. Georgeon, C. A. Goverde, G. N. Hatzopoulos, P. Gonczy, Y. D. Muller, G. Schwank, D. C. Swarts, A. J. Vecchio, B. L. Schneider, S. Ovchinnikov and B. E. Correia (2024). "BindCraft: one-shot design of functional protein binders." bioRxiv.Quijano-Rubio, A., Yeh, H.W., Park, J., Lee, H., Langan, R.A., Boyken, S.E., Lajoie, M.J., Cao, L., Chow, C.M., Miranda, M.C., Wi, J., Hong, H.J., Stewart, L., Oh, B.H., and Baker, D. (2021) De novo design of modular and tunable protein biosensors. Nature 591: 482-487.Rihtar, E., Lebar, T., Lainscek, D., Kores, K., Lesnik, S., Bren, U., and Jerala, R. (2023) Chemically inducible split protein regulators for mammalian cells. Nature chemical biology 19: 64-71.Saita, E., Abriata, L.A., Tsai, Y.T., Trajtenberg, F., Lemmin, T., Buschiazzo, A., Dal Peraro, M., de Mendoza, D., and Albanesi, D. (2015) A coiled coil switch mediates cold sensing by the thermosensory protein DesK. Mol Microbiol 98: 258-271.Scheller, L., M. Schmollack, A. Bertschi, M. Mansouri, P. Saxena and M. Fussenegger (2020). "Phosphoregulated orthogonal signal transduction in mammalian cells." Nat Commun 11(1): 3085.Shui, S., Gainza, P., Scheller, L., Yang, C., Kurumida, Y., Rosset, S., Georgeon, S., Di Roberto, R.B., Castellanos-Rueda, R., Reddy, S.T., and Correia, B.E. (2021) A rational blueprint for the design of chemically-controlled protein switches. Nat Commun 12: 5754.Silversmith, R.E. (2010) Auxiliary phosphatases in two-component signal transduction. Curr Opin Microbiol 13: 177-183.Siryaporn, A., and Goulian, M. (2008) Cross-talk suppression between the CpxA-CpxR and EnvZ-OmpR two-component systems in E. coli. Mol Microbiol 70: 494-506.Tebo, A. G. and A. Gautier (2019). "A split fluorescent reporter with rapid and reversible complementation." Nat Commun 10(1): 2822.Tinberg, C.E., Khare, S.D., Dou, J., Doyle, L., Nelson, J.W., Schena, A., Jankowski, W ., Kalodimos, C.G., Johnsson, K., Stoddard, B.L., and Baker, D. (2013) Computational design of ligand-binding proteins with high affinity and selectivity. Nature 501: 212- 216.Trajtenberg, F., M. Grana, N. Ruetalo, H. Both and A. Buschiazzo (2010). "Structural and enzymatic insights into the ATP binding and autophosphorylation mechanism of a sensor histidine kinase." J Biol Chem 285(32): 24892-24903.Trajtenberg, F., Imelio, J. A., Machado, M.R., Larrieux, N., Marti, M.A., Obal, G., Mechaly, A.E., and Buschiazzo, A. (2016) Regulation of signaling directionality revealed by 3D snapshots of a kinase:regulator complex in action. eLife 5.Vahedi-Faridi A, Eckey V, Scheffel F, Alings C, Landmesser H, Schneider E, Saenger W.Crystal structures and mutational analysis of the arginine-, lysine-, histidine-binding protein ArtJ from Geobacillus stearothermophilus. Implications for interactions of ArtJ with its cognate ATP-binding cassette transporter, Art(MP)2. J Mol Biol. 2008 Jan 11;375(2):448-59.Watson, J.L., Juergens, D., Bennett, N.R., Trippe, B.L., Yim, J., Eisenach, H.E., Ahern, W ., Borst, A. J., Ragotte, R.J., Milles, L.F., Wicky, B.I.M., Hanikel, N., Pellock, S.J., Courbet, A., Sheffler, W., Wang, J., Venkatesh, P., Sappington, I., Torres, S.V., Lauko, A., De Bortoli, V., Mathieu, E., Ovchinnikov, S., Barzilay, R., Jaakkola, T.S., DiMaio, F., Baek, M., and Baker, D. (2023) De novo design of protein structure and function with RF diffusion. Nature 620: 1089-1100.Wuichet, K., Cantwell, B.J., and Zhulin, I.B. (2010) Evolution and phyletic distribution of two-component signal transduction systems. Curr Opin Microbiol 13: 219-225.Zanghellini, A., L. Jiang, A. M. Wollacott, G. Cheng, J. Meiler, E. A. Althoff, D.Rothlisberger and D. Baker (2006). "New algorithms and an in silico benchmark for computational enzyme design." Protein Sci 15(12): 2785-2794.Zhang, Z., W. X. Shen, Q. Liu and M. Zitnik (2024). "Efficient generation of protein pockets with PocketGen." Nature Machine Intelligence 6(11): 1382-1395.

Claims

AMENDED CLAIMS received by the International Bureau on June 6, 2025 (06.06.2025)WE CLAIM:

1. A tunable engineered biosensor comprising: a chimeric dimer composed of a first protomer and a second protomer, each protomer comprising: a sensor module capable of recognizing an input signal; a histidine kinase (HK) catalytic module; and at least one heptad repeat sequence; wherein: at least one of the sensor module, heptad repeat sequence and or histidine kinase module is engineered; the at least one heptad repeat sequence of the first protomer and the second protomer are associate to form a coiled-coil structure; the at least one heptad repeat sequence is operably linked to the sensor module and to the histidine kinase module; the first protomer is dimerized with the second protomer into a closed conformation associated with a functional status; and the at least one coiled-coil structure is downstream of the sensor module; and wherein the dimer formed by the first protomer and second protomer adopts a closed conformation associated with the functional status or an open conformation associated with a different functional status; wherein, in the absence of a ligand, the coiled coil is oriented and stabilized in the closed conformation between protomers; and the ligand, when present, binds to the sensor module and a steric competition between the closed coiled-coil structure and the bound ligand-sensor module transitions the coiled-coil to the open conformation, and conformational coupling mediates signal transmission to the HK catalytic module; and wherein at least one mutation introduced in the HK catalytic module; and / or the sensor module and / or in the at least one heptad repeat of at least one of the protomer adjusts a trigger threshold; the trigger threshold being the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure; and upon reaching the trigger threshold, the at least one shift favors at least one transient open conformation of the dimer which induce a change in the functional status of the tunable chimeric biosensor.

2. The tunable biosensor of claim 1, wherein the HK catalytic module comprises a dimerization and histidine phosphotransfer (DHp) domain and an ATP binding domain (ABD) at the C-terminus.

3. The tunable biosensor of any one of claims 1 to 2, wherein the DHp domain comprises at least one heptad repeat.

4. A tunable engineered biosensor comprising: a dimer composed of a first protomer and a second protomer, each protomer comprising: a sensor module capable of recognizing an input signal; a helical linker comprising at least one coiled-coil structure including at least one heptad repeat sequence; and an effector module comprising: a small split effector fragment; a large split effector fragment; a flexible linker of at least 5 residues connecting the small split effector fragment and the large split effector fragment; wherein, the small split effector fragment and the large split effector fragment are capable of self-assembling into a functional module; the helical linker comprising at least one coiled-coil structure extending from the sensor module and the small split effector fragment; optionally, a dimerization module operably linked to the protomer to enhance or stabilize dimer formation between the first protomer and second protomer; and the first protomer dimerized with the second protomer in a closed conformation between protomers precluding the assembly of the small split effector fragment and large split effector fragment; wherein, in the absence of a ligand, the coiled coil is oriented and stabilized in the closed conformation; and the ligand, when present, binds to the sensor module and a steric competition between the closed coiled-coil structure and the bound ligand-sensor module transitions the coiled-coil to the open conformation, and conformational coupling mediates signal transmission to the effector fragment,the at least one shift favors at least one transient open conformation which exposes the small split effector fragment, thereby allowing self-assembly with large split effector fragment into the -effector module wherein, at least one mutation is introduced in the effector module, the sensor module, the helical linker and dimerization module to adjust a trigger threshold, the trigger threshold being the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure.

5. The tunable biosensor of claim 4, wherein the effector module is a luciferase comprising a small split luciferase fragment (SmBit) as the small split effector fragment connected to a large split luciferase fragment (LgBit) as the large split effector fragment by a flexible linker of at least 5 residues.

6. The tunable biosensor of claim 5, wherein at least one mutation is introduced in the luciferase module, the sensor module, or both, and / or the helical linker and / or dimerization module to adjust a trigger threshold, the trigger threshold being the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure.

7. A tunable engineered DNA binding biosensor comprising: a homodimer composed of two protomers, each protomer comprising: a sensor module capable of recognizing an input signal; a DNA binding domain; a helical linker comprising at least comprising at least one heptad repeat sequence; wherein, the sensor module is operably linked to the DNA binding domain through the helical linker; the heptad repeat sequence of the protomers are associated to form a coiled-coil structure; the coiled coil structure functionally links the sensor module to the DNA binding domain and is positioned downstream of the DNA domain; at least one mutation is introduced in the DNA binding domain and / or the sensor module and / or the helical linker adjusts a trigger threshold;the trigger threshold being the minimum amount of input signal required to transduce at least one allosteric change of the sensor module into at least one shift in the topology of the at least one coiled-coil structure; and in the absence of a ligand the coiled-coil is oriented and stabilized in the closed conformation, resulting in the stabilization of the two protomers, allowing for binding to the DNA sequence; and the ligand, when present, binds to the sensor module, and a steric competition between the coiled-coil structure and the bound ligand-sensor module transitions the coiled-coil to the open conformation, and the conformational coupling mediates signal transmission to the DNA binding domain, which disrupts the dimer interaction leading to the release of each protomer from the bound DNA target sequence.

8. The tunable biosensor of any one of claims 1 to 6, wherein the at least one coiled-coil structure extends from the catalytic module to the sensor module through a helical linker.

9. The tunable biosensor of any one of claims 1 to 3, wherein the helical linker is an S- helix.

10. The tunable biosensor of any one of claims 1 to 9, wherein the at least one coiled-coil structure comprises two or more heptad repeats.

11. The tunable biosensor of any one of claims 1 to 10, wherein the at least one heptad repeat deviates from the canonical sequence (abcdefg) with at least one of the core positions a and d occupied by a polar amino acid residue instead of a hydrophobic amino acid residue, the positions e or / and g being a polar or charge amino acid residue.

12. The tunable biosensor of anyone of claims 1 to 11, wherein the sensor module comprises a first sensor domain (domain A) and a second sensor domain (domain B) capable of assembling upon recognizing the input signal.

13. The tunable biosensor of anyone of claims 1 to 11, wherein the sensor module comprises a first sensor domain (domain A) and a second sensor domain (domain B) capable of disassembling upon recognizing the input signal.

14. The tunable biosensor of claim 12, wherein the first sensor domain is either connected to the second sensor domain by a flexible linker or is not covalently linked to the sensor second domain.

15. The tunable biosensor of any of claims 12 to 13, wherein the first sensor domain and the second sensor domain comprise split fragments of a ligand-binding site of a protein, the ligand binding site recognizing the input signal.

16. The tunable biosensor of any of claims 12 to 13, wherein the first sensor domain and the second sensor domain are derived from distinct proteins.

17. The tunable biosensor of claim 13, wherein the first sensor domain is absent, and the second sensor domain recognize a macromolecule.

18. The tunable biosensor of any of claims 1 to 3 and 7 to 15, wherein the sensor module and the HK catalytic module are at the N-terminus and the C-terminus of each protomer, respectively.

19. The tunable biosensor of any of claims 1 to 17, wherein the dimer is in soluble form or membrane bound.

20. The tunable biosensor of claim 19, wherein the membrane-bound dimer comprises at least one transmembrane domain (TD).

21. The tunable biosensor of claim 20, wherein a first TD is configured at the C-terminus of the sensor module.

22. The tunable biosensor of any one of claims 20 to 21, wherein a second TD or a signal peptide is configured either at the N-terminus or C-terminus of the sensor module.

23. The tunable biosensor of claim 22, wherein the second TD or the signal peptide is connected to the second sensor domain or the first sensor domain by a flexible linker.

24. The tunable biosensor of claim 23, wherein the flexible linker comprises either a cleavage site or has a length of at least 10 amino acid residues.

25. The tunable biosensor of any one of claims 1 to 24, wherein the trigger threshold is decreased by introducing at least one mutation destabilizing the closed conformation to favorthe transient open conformation which induces a change in the functional status of the tunable biosensor.

26. The tunable biosensor of claim 25, wherein the at least one destabilizing mutation: destabilizes the at least one coiled-coil structure, and / or increases the binding affinity of the sensor module for its input signal, and / or favors interactions between the first sensor domain and the second sensor domain of the sensor module, and / or disrupts interactions between the DHp domain and the ADB domain, and / or destabilizes interactions between the transmembrane domains of the dimer.

27. The tunable biosensor of any one of claims 1 to 26, wherein the trigger threshold is increased by introducing at least one mutation stabilizing the closed conformation associated with a functional status characterized by an active or inactive auto-kinase and / or an active or inactive phosphatase.

28. The tunable biosensor of claim 27, wherein the at least one stabilizing mutation: stabilizes the at least one coiled-coil structure, and / or decreases the binding affinity of the sensor module for its input signal, and / or disrupts interactions between the first sensor domain and the second sensor domain of the sensor module, and / or favors interactions between the DHp domain and the ADB domain, and / or stabilizes transmembrane interactions.

29. The tunable biosensor of any one of claims 1 to 3 and 8 to 28, wherein the functional status of the closed conformation is characterized by an auto-kinase activity, and wherein the at least one mutation destabilizing the closed conformation favors rearrangement to the open conformation and induces the inactivation of the auto-kinase resulting in the inactivation of the biosensor.

30. The tunable biosensor of claim 20, wherein the functional status of the open conformation is characterized by a phosphatase activity.

31. The tunable biosensor of claim 29 or 30, wherein the functional state of the closed conformation is characterized by a phosphatase activity, andthe at least one mutation destabilizing the closed conformation favors rearrangement to the open conformation which induces the inactivation of the phosphatase.

32. The tunable biosensor of claim 31, wherein the functional status of the open conformation is characterized by an auto-kinase activity.

33. The tunable biosensor of any one of claims 1 to 3 and 8 to 26, wherein the functional state of the closed conformation is characterized by an inactive auto-kinase activity, and wherein the at least one mutation destabilizing the closed conformation favors rearrangement to the open conformation which induces the activation of the auto-kinase, resulting in the activation of the biosensor.

34. The tunable biosensor of claim 34, wherein the trigger threshold is increased by introducing at least one mutation stabilizing the closed conformation associated with a functional status characterized by an active or inactive auto-kinase and / or an active or inactive phosphatase.

35. A two-component system (TCS) comprising: a biosensor according to any of claims 1 to 32, and a cognate response regulator (RR) comprising a receiver domain, wherein the activated biosensor transfers a phosphoryl moiety to a phosphorylation site of the cognate RR.

36. The TCS of claim 35, wherein the receiver domain of RR is a DNA binding domain.

37. A method to modulate expression of a target gene, comprising exposing the target gene to the TCS of claim 35 or claim 36, resulting in RR phosphorylation, induced dimerization and binding to the promotor of the target gene.

38. A biocomputing system comprising a plurality of TCSs according to claim 35 or claim 36, each TCS comprising a distinct biosensor and its cognate RR, wherein the plurality of TCSs can be connected through phosphorylation and / or dephosphorylation reactions to integrate and process information.

39. A procaryotic cell comprising the TCS of claim 35 or claim 36 or the biocomputing system of claim 38 or the tunable biosensor according to any one of claims 1 to 33.

40. A eukaryotic cell comprising the TCS of claim 35 or claim to 36.

41. An in silico method for designing a tunable dimeric biosensor comprising a sensor module and an effector module, comprising: generating a sensor module, the sensor module comprising two sensor domains, domain A and domain B, capable of assembling to form a ligand binding site in a closed conformation upon recognizing the ligand, comprising: identifying a ligand binding site, separating key residues of the ligand binding site into a first set of ligand binding residues and a second non-overlapping set of ligand binding residues; embedding the first set of ligand binding residues and a helical segment within the domain B designed by partial diffusion or inpainting to allow the folding of the first set of ligand binding residues and the helical segment into a functional three- dimensional structure, wherein the helical segment comprising at least one coiled-coil structure placed at the C-terminus of domain B, is positioned to favor the formation of a dimer in a closed conformation, and wherein the first set of the ligand binding residues is oriented towards the dimer interface upon dimerization; embedding the second non-overlapping set of ligand binding residues in domain A designed by partial diffusion or inpainting to allow the folding of the second nonoverlapping set of ligand binding residues into a functional three-dimensional conformation structure, wherein the first set of ligand binding residues and the second non-overlapping set of ligand binding residues form the functional ligand binding site capable of accommodating the ligand, and wherein domain A and domain B assemble into the functional sensor module capable of adopting an open or closed conformation; wherein the sensor module is operably linked to the effector module through a helical linker comprising at least one heptad repeat.

42. The method of claim 41, wherein the ligand binding site is identified using available structures and / or using a molecular docking algorithm and / or artificial intelligence algorithms.