Boolean logic-gated protein presentation via autonomously compiled molecular topology

Abstract

Biomaterials and polypeptide-based biological circuits configured for conditional release of a cargo therefrom. Logical biological circuit elements, including YES, AND, and OR elements, can be used to construct biosensors and implantable devices that are configured to conditionally release a biologically relevant cargo therefrom, such as a drug, biotherapeutic, or biomarker, in an appropriate scenario.

Description

BOOLEAN LOGIC-GATED PROTEIN PRESENTATION VIA AUTONOMOUSLY COMPILED MOLECULAR TOPOLOGY CROSS-REFERENCE(S) TO RELATED APPLICATION(S)

[0001] This application claims the benefit of, and priority to, U.S. Provisional Application No.63 / 654,359 filed May 31, 2024; the disclosure of which is incorporated by reference herein in its entirety for all purposes. STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the sequence listing is 3915-P1351WO.UW-Sequence- Listing.xml. The XML file is 83,826 bytes; was created on May 21, 2025; and is being submitted electronically via Patent Center with the filing of the specification. STATEMENT OF GOVERNMENT LICENSE RIGHTS

[0003] This invention was made with government support under Grant No. R35GM138036, awarded by the National Institutes of Health (NIH), and Grant No. 1807398, awarded by the National Science Foundation (NSF). The government has certain rights in the invention. BACKGROUND

[0004] Programming stimuli-responsiveness into biomaterials and biohybrid constructs represents an exciting frontier poised to galvanize many advanced bioengineering applications. For example, controlled presentation of biomolecules (e.g., proteins, peptides, polysaccharides, nucleic acids) in / from materials holds direct applicability in therapeutic delivery, tissue engineering, organoid development, and the biomanufacturing of value-added chemicals.

[0005] Co-opting novel methods from organic- and synthetic bio-based chemistries, stimuli-responsive material platforms have previously been reported that sense and act upon diverse inputs including light, redox potential, enzyme, and pH. While these prior approaches have transformed biomaterials from static to dynamic systems, most of3915-P1351WO.UW -1-these prior platforms have exhibited only single-input / single-output-type responses, and this has dramatically limited their scope of use.

[0006] As such, there is a significant need for biomaterials and associated products that exhibit multi-input / single-output and multi-input / multi-output responses for controlled presentation of biomolecules and applications in biosensing, medical monitoring, environmental monitoring, and other fields. The present disclosure addresses these and other long-felt and unmet needs in the art. SUMMARY

[0007] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0008] In an aspect, the disclosure provides a biomaterial comprising a biological circuit configured for a conditional release of a cargo from an immobilized portion of the biomaterial, the biological circuit comprising: a polypeptide comprising a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; and wherein one or more protease sites is / are proteolytically cleaved, the conditional release occurs for release of the cargo from the biomaterial.

[0009] In embodiments, the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0010] In embodiments, the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0011] In embodiments, the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the3915-P1351WO.UW -2-cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0012] In embodiments, the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0013] In embodiments, the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0014] In embodiments, the biological circuit comprises a 5-input-responsive [AND / (OR)]OR(AND) gate.

[0015] In embodiments, the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

[0016] In embodiments, the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

[0017] In embodiments, the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.

[0018] In embodiments, the tadpole motif, the n-armed star motif, and / or the H- shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

[0019] In embodiments, an immobilized portion of the biomaterial comprises a solid support and the conditional release occurs for release of the cargo from the solid support.3915-P1351WO.UW -3-

[0020] In embodiments, a biomaterial comprises a liquid matrix that comprises a biological agent configured to bind to the cargo.

[0021] In embodiments, a biological agent is a cell or a portion thereof that comprises an antigen, and wherein the cargo is an antibody or a portion thereof that comprises an antigen-binding domain.

[0022] In another aspect, the disclosure provides a biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising a biomaterial.

[0023] In another aspect, the disclosure provides a polypeptide, comprising: a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; and a cargo, wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; wherein one or more protease sites is / are proteolytically cleaved, a conditional release occurs for release of the cargo from the polypeptide.

[0024] In embodiments, the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0025] In embodiments, the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0026] In embodiments, the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0027] In embodiments, the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically3915-P1351WO.UW -4-cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0028] In embodiments, the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0029] In embodiments, the biological circuit comprises a 5-input-responsive [AND / (OR)]OR(AND) gate.

[0030] In embodiments, the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

[0031] In embodiments, the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

[0032] In embodiments, the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.

[0033] In embodiments, the tadpole motif, the n-armed star motif, and / or the H- shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

[0034] In another aspect, the disclosure provides a nucleic acid comprising a polynucleotide sequence encoding a polypeptide.

[0035] In embodiments, the polynucleotide sequence comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identity with at least one sequence, or at least one portion of a sequence, selected from the group consisting of: SEQ ID NOs:1-37.

[0036] In another aspect, the disclosure provides an expression cassette and / or expression vector comprising a polynucleotide sequence that is configured for expression of a polypeptide in a biological system.3915-P1351WO.UW -5-

[0037] In another aspect, the disclosure provides a genetically modified host cell comprising a nucleic acid.

[0038] In another aspect, the disclosure provides a host organism comprising a genetically modified host cell.

[0039] In another aspect, the disclosure provides a biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising a genetically modified host cell. DESCRIPTION OF THE DRAWINGS

[0040] The foregoing aspects and many of the attendant advantages of this disclosure will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings.

[0041] FIGs 1A-1G show an example of how autonomously compiled molecular topologies enable Boolean logic-based protein presentation, according to aspects of the disclosure. FIG.1A, The YES-gate tether contains a single protease-responsive sequence (rectangle). Introduction of the cognate protease cleaves this sequence, breaking the covalent linkage between the protein cargo (star) and tethering motif (semicircle). FIG. 1B, The OR-gate tether contains two protease-responsive sequences (left and right rectangles) connected in series. The introduction of either relevant protease cleaves the sequence, resulting in cargo release. FIG.1C, The AND-gate tether contains two protease- responsive sequences (left and right rectangles) connected in parallel. The introduction of one cognate protease cleaves one arm but does not fully sever the cycle. Simultaneous introduction of both proteases cleaves both arms, resulting in protein release. FIG. 1D, YES / OR / AND operators can be hierarchically layered to engineer higher-order logical responses. Seventeen unique protein tethering topologies can be obtained through combination of the three base operations with three distinct actuator-proteases. Each region of the Venn diagrams displayed represents a unique combination of protease inputs and indicates whether the tether is predicted to cleave (shaded) or remain intact (non-shaded). FIG. 1E, SICLOPPS provides a genetically encoded and near-traceless approach for the N-to-C cyclization of proteins based on intein trans-splicing. FIG. 1F, Tag / Catcher chemistries form isopeptide bonds between complementary protein modules via SpyLigation, SnoopLigation, and DogLigation. FIG. 1G, Reactions depicting protease3915-P1351WO.UW -6-recognition of target sequence and subsequent cleavage: the recognition sequence ENLYFQ↓S is recognized and cleaved by the TEV protease; LAET↓G by eSrt(2A9); LPES↓G by eSrtA(4S9).

[0042] FIGs 2A-2I show an example of how autonomously assembled logic operators undergo pre-programmed cleavage from bead surfaces in response to actuator input combinations, according to aspects of the disclosure. FIG. 2A, SpyCatcher- functionalized magnetic beads can be decorated with different logical operators holding a SpyTag motif through SpyLigation. In this example, mGreenLantern-A is cleaved from the resin and solubilized in response to the A treatment. FIG. 2B, Supernatant fluorescence indicates logic-based release, here to input A. FIG.2C, The response profiles of the YES- gated single-input tethers. FIGs 2D-2E, The response profiles of the two-input (FIG.2D) OR-gated and (FIG. 2E) AND-gated tethers. FIGs 2F-2I, The response profiles of the three-input (FIG.2F) OR / (AND)-, (FIG.2G) AND / (OR)-, (FIG.2H) OR / OR-, and (FIG. 2I) AND / AND-gated tethers. Plot titles correspond to the protein tether identity; the y-axis represents extent of protein release from bead surfaces as measured via supernatant fluorescence; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(2S9), and T indicates TEV. The AST bar indicates a condition expected to yield tether cleavage, whereas the other bars (N, A, S, T, AS, AT, ST) indicate conditions not expected to yield release. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0043] FIGs 3A-3G show example SDS-PAGE gels for 17 mGreenLantern logical constructs following in-solution treatment, according to aspects of the disclosure. Following in-solution treatment of each of the 17 YES / OR / AND gated mGreenLantern species with A, S, and T, samples were analyzed via SDS-PAGE. Representative uncropped gels for each are shown as well as numbered protein stickers to label all possible cleavage products. FIG. 3A) YES-gated single-input tethers. FIGs 3B-3C) Two-input (FIG. 3B) OR-gated and (FIG. 3C) AND-gated tethers. FIGs 3D-3G) Three-input (FIG. 3D) OR / (AND)-, (FIG.3E) AND / (OR)-, (FIG.3F) OR / OR-, and (FIG.3G) AND / AND-gated tethers. Image titles correspond to the protein tether identity. For each image, lanes from left to right correspond to: ladder,N,A,S,T,AS,AT,ST,AST;Nindicates no treatment,Aindicates eSrtA(2A9), S indicates eSrtA(2S9), and T indicates TEV. Note that the high molecular weight band present in each input combination that utilizes T is the TEV protease3915-P1351WO.UW -7-(MW 71.2 kDa). From top to bottom, ladder bands correspond to proteins with molecular weights of 180, 130, 100, 70, 55, 40, 35, 25, and 15 kDa.

[0044] FIGs 4A-4H show examples of SDS-PAGE analysis of in-solution treatment of autonomously compiled mGreenLantern pendants, according to aspects of the disclosure. FIG. 4A, mGreenLantern-A∧(S∨T) is differentially cleaved following the 8 possible input combinations of A, S, and T. Input conditions A, S, T, and ST are expected to linearize the product but not induce payload release, leading to a product with less electrophoretic mobility than the initial cyclic construct. Payload release is expected following input conditions AS, AT, and AST, accompanied with a product band that migrates further than the starting species. FIG.4B, Changes in topology and / or molecular weight in response to a specific set of inputs leads to changes in protein electrophoretic mobility, which can be analyzed through gel densitometry. FIG. 4C, The response profiles of the YES-gated single-input tethers. FIGs 4D-4E, The response profiles of the two-input (FIG. 4D) OR-gated and (FIG.4E) AND-gated tethers. FIGs 4F-4H, The response profiles of the three-input (FIG. 4F) OR / (AND)-, (FIG. 4G) AND / (OR)-, and (FIG. 4H) OR / OR- gated tethers. Plot titles correspond to the protein tether identity; the y-axis represents extent of cleavage as measured through migration on an SDS-PAGE with gel densitometry analysis; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9),Sindicates eSrtA(2S9), andTindicates TEV. Shaded bars indicate conditions expected to yield tether cleavage or expected to keep the tether non-cleaved. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0045] FIGs 5A-5C show example results of SDS-PAGE gels of mCherry-S, mCerulean-T, and mCerulean-A∧T following in-solution treatment, according to aspects of the disclosure. Following in-solution treatment of mCherry-S, mCerulean-T, and mCerulean-A∧T each with A, S, and T, samples were analyzed via SDS-PAGE. Representative uncropped gels for each are given here. FIG.5A) mCherry-S, note a second band for the native protein is present due to chromophore separation during sample preparation, characteristic of mCherry, (FIG. 5B) mCerulean-T, and (FIG. 5C) mCerulean-A∧T, note a second band for the native protein due to the presence of higher- order macrocycles (less than 10% of product). Image titles correspond to the protein identity. For each image, lanes from left to right correspond to: ladder, N, A, S, T, AS, AT, ST, AST; N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(2S9), and T3915-P1351WO.UW -8-indicates TEV. Note that the high molecular weight band present in each input combination that utilizes T is the TEV protease (MW 71.2 kDa). From top to bottom, ladder bands correspond to proteins with molecular weights of 180, 130, 100, 70, 55, 40, 35, 25, and 15 kDa.

[0046] FIGs 6A-6C show example results from SDS-PAGE analysis of in- solution treatment of autonomously compiled mCherry and mCerulean pendants, according to aspects of the disclosure. FIGs 6A-6C, Changes in topology and / or molecular weight in response to a specific set of inputs leads to changes in protein electrophoretic mobility, which can be analyzed through gel densitometry. Results are shown for species (FIG.6A) mCherry-S, (FIG. 6B) mCerulean-T, and (FIG. 6C) mCerulean-S∧T. Plot titles correspond to the protein tether identity; the y-axis represents extent of cleavage as measured through migration on an SDS-PAGE with gel densitometry analysis; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(2S9), and T indicates TEV. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0047] FIGs 7A-7C show examples of how autonomously compiled molecular topologies enable the multiplexed and independently triggered release of distinct proteins from a hydrogel network, according to aspects of the disclosure. FIG.7A, PEG-tetraBCN, TEG-diazide, and SpyCatcher-azide ligated to SpyTagged proteins of interest react via SPAAC to form functionalized hydrogels. FIG.7B, mGreenLantern-A∨T, mCherry-S, and mCerulean-S∧T are tethered homogenously into an underlying PEG network, each exhibiting a different logic-based release profile. FIG.7C, Appropriate protein is released when the correct input combination is present. Shaded bars (mGreenLantern, mCherry, mCerulean) indicate conditions expected to yield protein release, whereas opaque bars denote conditions not expected to result in release. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0048] FIGs 8A-8B show an example of multiplexed YES-gated release of proteins from hydrogel biomaterials, according to aspects of the disclosure. FIG. 8A, mGreenLantern-A, mCherry-S, and mCerulean-T are tethered homogenously into an underlying PEG-based hydrogel network via SpyLigation, each exhibiting a different YES- gated response. FIG. 8B, Appropriate proteins are individually released when their corresponding input is present. Shaded bars (left for mGreenLantern, center for mCherry, right for mCerulean, within each triplicate bar grouping) indicate conditions expected to3915-P1351WO.UW -9-yield protein release, whereas opaque bars denote conditions not expected to result in release. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0049] FIGs 9A-9D show genetic encodability enables seamless scale-up in logical response complexity, according to aspects of the disclosure. FIG. 9A, mGreenLantern-[(A∨S)∧T]∨(C∧V) can be assembled on one expression frame and purified rapidly. Each region of the Venn diagrams displayed represents a unique combination of protease inputs and indicates whether the tether is predicted to cleave (shaded) or remain intact (non-shaded). FIG.9B, Reactions depicting protease recognition of target sequence and subsequent cleavage: the recognition sequence ENLYFQ↓S is recognized and cleaved by TEV; LAET↓G by eSrt(2A9); LPES↓G by eSrtA(4S9); LEVLFQ↓GP by HRV-3C; ETVRFQ↓S by TVMV. FIG. 9C, mGreenLantern- [(A∨S)∧T]∨(C∧V) is readily purified and obtained with the correct molecular mass. FIG. 9D, The response profiles of the 5-input system. The y-axis represents extent of protein release from bead surfaces as measured via supernatant fluorescence; the x-axis indicates treatment conditions, wherein N indicates no treatment, A indicates eSrtA(2A9), S indicates eSrtA(2S9), T indicates TEV, V indicates TVMV, and C indicates HRV-3C. Shaded bars indicate conditions expected to yield tether cleavage or to keep the tether uncleaved. Error bars correspond to ±1 standard deviation about the mean with propagated uncertainties for n=3 experimental replicates.

[0050] FIG. 10 shows example results from SDS-PAGE analysis of purified mGreenLantern-[(A∨S)∧T]∨(C∧V), according to aspects of the disclosure. SDS-PAGE analysis of estimated mass and protein purity of mGreenLantern-[(A∨S)∧T]∨(C∧V). The expected molecular weight is 53,946 kDa. The single band around 55 kDa on the SDS- PAGE gel confirms successful expression and purification of the desired species.

[0051] FIGs 11A-11C show example Boolean logic-based labeling of the mammalian extracellular membrane, according to aspects of the disclosure. FIG. 11A, HER2-expressing human breast cancer cells are tagged with an HER2Nb-C∧(A∨T)-eGFP, localizing a fluorophore to the extracellular membrane via a nanobody-receptor interaction. An AND / (OR)-gated response to eSrtA(2A9) (A), TEV (T), and HRV-3C (C) prevents membrane labeling. FIG. 11B, Fluorescent confocal microscopy indicates expected membrane labeling with eGFP (perinuclear shading), with nuclei counterstained with Hoechst 33342 (nuclear shading). Scale bar = 20 µm. FIG.11C, Logically specified cell3915-P1351WO.UW -10-labeling was further confirmed by flow cytometry. CT, AC, and ACT are conditions in which release is expected (i.e., no cell labeling with eGFP); N, A, C, T and AT are conditions not expected to yield release.

[0052] FIGs 12A-12D show an example of autonomous compilation and logic- defined localization within mammalian cells, according to aspects of the disclosure. FIG. 12A, HEK293 cells are stably modified via lentiviral transduction to express an mGreenLantern-C∨(T∧V)-CAAX fusion that translocates to the intracellular plasma membrane. When cells are subsequently transfected with plasmids encoding for each of the 8 relevant protease combinations, the fluorescent species is liberated from the cell membrane into the cytosol following an OR / (AND)-gated response to HRV-3C (C), TEV (T), and TVMV (V). FIG. 12B, Fluorescent confocal microscopy indicates expected membrane labeling with mGreenLantern following treatment. Scale bar = 10 µm. FIGs 12C-12D, CellProfiler-based analysis of mGreenLantern intracellular positioning illustrates expected cytosolic presentation followingC,CT,CV,TV, andCTVtreatments. N, C, V, CT, CV, TV, and CTV are conditions in which release is expected (i.e., cytosolic mGreenLantern); T is a condition not expected to yield release.

[0053] FIGs 13A-13F show example results from kinetic analysis for protease- variable YES-gate cleavage, according to aspects of the disclosure. mGreenLantern-A, mCherry-S, and mCerulean-T were treated at 4 °C with their respective inputs [i.e., eSrtA(2A9), eSrtA(4S9), and TEV] at differing molar ratios of protease:construct (1:500, 1:200 and 1:50 for A, S; 1:50, 1:20 and 1:5 for T) and sampled over time prior to SDS-PAGE and gel densitometry analysis. Representative uncropped gels for each are given here. Analysis of percent of logical operator cleaved for each tested molar ratio over time for (FIG. 13A) mGreenLantern-A, (FIG. 13B) mCherry-S, and (FIG. 13C) mCerulean-T. SDS-PAGE time course of each tested molar ratio for (FIG. 13D) mGreenLantern-A, (FIG.13E) mCherry-S, and (FIG.13F) mCerulean-T. For each image, lanes from left to right correspond to: ladder, 0 minutes, 15 minutes, 1 hour, 3 hours, and 6 hours. From top to bottom, ladder bands correspond to proteins with molecular weights of 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa. Each data point represents the mean with error bars corresponding to ±1 standard deviation, n=3 experimental replicates.

[0054] FIGs 14A-14F show example results from kinetic analysis for temperature-variable YES-gate cleavage, according to aspects of the disclosure. mGreenLantern-A, mCherry-S, and mCerulean-T were treated with their respective inputs3915-P1351WO.UW -11-[i.e., eSrtA(2A9), eSrtA(4S9), and TEV] at varying temperatures (4 °C, 25 °C, and 37 °C), then sampled over time prior to SDS-PAGE and gel densitometry analysis. Protease:construct ratios were held at 1:100 forAandS, and 1:10 forT. Representative uncropped gels for each are given here. Analysis of percent of logical operator cleaved for each tested temperature over time for (FIG.14A) mGreenLantern-A, (FIG.14B) mCherry- S, and (FIG. 14C) mCerulean-T. SDS-PAGE time course of each tested molar ratio for (FIG.14D) mGreenLantern-A, (FIG.14E) mCherry-S, and (FIG.14F) mCerulean-T. For each image, lanes from left to right correspond to: ladder, 0 minutes, 15 minutes, 1 hour, 3 hours, and 6 hours. From top to bottom, ladder bands correspond to proteins with molecular weights of 250, 150, 100, 75, 50, 37, 25, 20, 15 and 10 kDa. Each data point represents the mean with error bars corresponding to ±1 standard deviation, n=3 experimental replicates.

[0055] FIGs 15A-15G show example SDS-PAGE gels for 17 mGreenLantern logical constructs following in-solution treatment, according to aspects of the disclosure. Following in-solution treatment of each of the 17 YES / OR / AND gated mGreenLantern species withA,S, andT, samples were analyzed via SDS-PAGE. Representative uncropped gels for each are given here as well as numbered protein stickers to label all possible cleavage products. FIG.15A) YES-gated single-input tethers. FIGs 15B-15C) Two-input (FIG. 15B) OR-gated and (FIG. 15C) AND-gated tethers. FIGs 15D-15G) Three-input (FIG.15D) OR / (AND)-, (FIG.15E) AND / (OR)-, (FIG.15F) OR / OR-, and (FIG.15G) AND / AND-gated tethers. Image titles correspond to the protein tether identity. For each image, lanes from left to right correspond to: ladder, N, A, S, T, AS, AT, ST, AST; N indicates no treatment,Aindicates eSrtA(2A9),Sindicates eSrtA(2S9), andTindicates TEV. Note that the high molecular weight band present in each input combination that utilizesTis the TEV protease (MW 71.2 kDa). From top to bottom, ladder bands correspond to proteins with molecular weights of 180, 130, 100, 70, 55, 40, 35, 25, and 15 kDa.

[0056] FIGs 16A-16C show example SDS-PAGE gels of mCherry-S, mCerulean-T, and mCerulean-A∧T following in-solution treatment, according to aspects of the disclosure. Following in-solution treatment of mCherry-S, mCerulean-T, and mCerulean-A∧T each with A, S, and T, samples were analyzed via SDS-PAGE. Representative uncropped gels for each are given here. FIG. 16A) mCherry-S, note a second band for the native protein is present due to chromophore separation during sample preparation, characteristic of mCherry, (FIG. 16B) mCerulean-T, and (FIG. 16C) mCerulean-A∧T, note a second band for the native protein due to the presence of higher-3915-P1351WO.UW -12-order macrocycles (less than 10% of product). Image titles correspond to the protein identity. For each image, lanes from left to right correspond to: ladder,N,A,S,T,AS,AT,ST,AST;Nindicates no treatment,Aindicates eSrtA(2A9),Sindicates eSrtA(2S9), andTindicates TEV. Note that the high molecular weight band present in each input combination that utilizes T is the TEV protease (MW 71.2 kDa). From top to bottom, ladder bands correspond to proteins with molecular weights of 180, 130, 100, 70, 55, 40, 35, 25, and 15 kDa.

[0057] FIG. 17 shows an example truth table for mGreenLantern- [(A∨S)∧T]∨(C∧V) release, according to aspects of the disclosure. The figure shows a Boolean logic-based truth table for the expected release of mGreenLantern- [(A∨S)∧T]∨(C∧V).

[0058] FIGs 18A-18B shows example results that protease conditions are biocompatible with mammalian cells, according to aspects of the disclosure. FIG. 18A, Confocal images of HEK293T post treatment with either 1mM DTT, 1mM CaCl2, 18mM GGG, 1µM of eitherA,S, orT, a combination of all conditions, no supplements (positive control) or 50% DMSO (negative control). The optical channels correspond with the Live stain Calcein AM, or EtHD-1. Scale bar = 500 µm. FIG.18B, Percentage of live cells for each treatment group. Error bars correspond to the mean with ±1 standard deviation, n=3 experimental replicates.

[0059] FIG.19 shows an example diagram illustrating SpyLigation, according to aspects of the disclosure.

[0060] FIG. 20 shows example results from SDS-PAGE gel analysis of HER2Nb-C∧(A∨T)-eGFP, according to aspects of the disclosure. SDS-PAGE analysis for reaction product [HER2Nb-C∧(A∨T)-eGFP] following ligation of HER2Nb-C∧(A∨T)- SpyTag to eGFP-SpyCatcher. The molecular weight of the ligated conjugate is 65.3 kDa. The band between 50 kDa to 75 kDa corresponds to the ligated species. Some amount of unreacted eGFP-SpyCatcher (42.2 kDa) remained in the sample. Since the eGFP- SpyCatcher was not expected to alter membrane binding studies, no additional purification was performed.

[0061] FIGs 21A-21B show example results from in-solution analysis of HER2Nb-C∧(A∨T)-eGFP logical response, according to aspects of the disclosure. FIG. 21A, Normalized cleavage of HER2Nb-C∧(A∨T)-eGFP, after treatment with all relevant combinations of protease inputs (A, C, T). Complete cleavage separating HER2Nb and the3915-P1351WO.UW -13-eGFP proteins was expected for input conditions AC, TC, and ATC. Release was determined via gel densitometry following analysis by SDS-PAGE. FIG. 21B, Coomassie-stained SDS-PAGE of HER2Nb-C∧(A∨T)-eGFP treated with all relevant protease combinations (lane order from left to right: ladder, N, A, C, T, AC, AT, TC, ATC). Associated protein structures are included to depict expected post-treatment fragments. The band at 42.2 kDa in all samples corresponds to eGFP-SpyCatcher. The band between 35-40 kDa for all conditions that containCis HRV-3C. The three low molecular weight bands between 15-25 kDa are the cleaved HER2Nb fragment (slight shift is observed due to remaining attached logical operation). From top to bottom, ladder bands correspond to proteins with molecular weights of 180, 130, 100, 70, 55, 40, 35, 25, 15 and 10 kDa.

[0062] FIG. 22 shows example results that HER2Nb-C∧(A∨T)-eGFPspecifically labels HER2+cells, according to aspects of the disclosure. SK-BR-3 andHEK293 cells were treated with HER2Nb-C∧(A∨T)-eGFP and nuclei counterstained with Hoechst 33342. The HER2+SK-BR-3 cells exhibited expected membrane labeling, as visualized by fluorescent confocal microscopy. Scale bar = 200 µm.

[0063] FIG. 23 shows an example flow cytometry gating strategy, according to aspects of the disclosure. The figure shows a gating strategy for flow cytometry for SK- BR3 cells treated with HER2Nb-C∧(A∨T)-eGFP. Cells were first gated to include only live cells (FSC-A vs. SSC-A). This cell population was then gated to remove doublets (FSC-A vs. FSC-H). Further, the cell population was then analyzed for the percent of positive eGFP labeled cells. The eGFP+threshold was 1.5 x 103AFU. This representative gating strategy shows the cell population for proteaseA-treated HER2Nb-C∧(A∨T)-eGFP, expected to be positive for eGFP. DETAILED DESCRIPTION

[0064] The present disclosure provides novel, innovative approaches that enable the design and use of biomaterials that are “programmed” to conditionally release a cargo therefrom in response to one or more environmental conditions or stimuli. The examples disclosed herein make use of protease recognition sites, engineered within polypeptides, that are recognized and cleaved by corresponding proteases for the physical release of one or more portions of a polypeptide that are connected to (or that otherwise interfere with) release of the cargo from an immobile substrate of the biomaterial. The polypeptide can be linear and / or branched, or non-linear, for the creation of a polypeptide-based biological3915-P1351WO.UW -14-circuit having parallel, non-parallel, redundant, orthogonal, and / or non-orthogonal inputs for the controlled and conditional release of the cargo therefrom, according to design choice. Biomaterials can be implemented into any form factor and commercial embodiment according to need, including but not limited to: biosensors, implantable devices, environmental monitoring apparatuses, drug sensing and delivery devices, and the like.

[0065] As such, the disclosure provides practical implementations that reduce to practice the broader concept of controlled release of a cargo within the context of a stationary or confined biomaterial or biological matrix, including but not limited to artificial and non-living biomaterials as well as organic and living biomaterials such as cells, tissues, and organisms.

[0066] In addition, while the disclosed proof-of-concept examples provide practical approaches for building layered and complex biological circuits for controlled release of the cargo, the disclosed approaches can be combined with other molecular biology approaches, including gene regulation schemes for controlled expression of one or more proteases based on one or more environmental stimuli, which then operate on one or more Boolean logic-gated polypeptides of the disclosure. Such environmental stimuli can correspond to changes in a characteristic of a fluid being monitored by a biosensor of the disclosure, changes in drug levels in a patient in need of additional drug delivery thereto, changes in blood sugar in a patient in need of additional or emergency insulin delivery, and the like.

[0067] Accordingly, the disclosed approaches, while immediately practical as evidenced by the present disclosure, are also intended as being able to be combined with other environmental monitoring and gene regulation schemes and are additionally practical for at least this feature. Boolean Logic-Gated Polypeptides

[0068] Boolean logic-gated polypeptides of the disclosure are configured for a rearrangement that occurs with one or more conditions being met. An example polypeptide includes one or more protease recognition sites for conditional cleavage of the polypeptides in the presence of one or more corresponding proteases, such that the one or more proteases being present corresponds to, or results from, the one or more conditions being met. The polypeptides can be linked to the cargo, directly or indirectly, by one or more peptide bonds and / or one or more other covalent bonds, or can be complexed with the cargo such as by3915-P1351WO.UW -15-way of one or more non-covalent bonds or interactions. In various implementations, the use of polypeptides that are encodable by DNA enables robust and reliable synthesis of complex and scalable biological circuits for programmable, conditional release of cargo according to the design of the biological circuit.

[0069] Accordingly, in an aspect, the disclosure provides a polypeptide comprising a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide.

[0070] Depending on the physical arrangement of the first and second protease sites relative to each other on the polypeptide (which may be branched or linear / unbranched), release of a cargo from the polypeptide may require cleavage of only one of the first and second protease sites (e.g., an “OR” gate) or may require cleavage of both the first and second protease sites (e.g., an “AND” gate). The cargo can be directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof, in various embodiments. As one or more protease sites is / are proteolytically cleaved, a conditional release occurs for release of the cargo from the polypeptide.

[0071] As such, in various embodiments, the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial. In these and other embodiments, the conditional release can comprise an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0072] In various embodiments, an AND gate is comprised of at least two “YES” gates, arranged in parallel, such that release requires cleavage of both YES gates (each YES gate of the AND gate comprises a protease cleavage site). An OR gate is comprised of at least two “YES” gates, arranged in series, such that release requires cleavage of either YES gate but does not require cleavage of both YES gates (each YES gate of the OR gate comprises a protease cleavage site).

[0073] In embodiments, the biological circuit comprises an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur (i.e., for the3915-P1351WO.UW -16-“AND” condition of the two “YES” gates to be met) for release of the cargo from the immobilized portion of the biomaterial, and in embodiments, comprises an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0074] In embodiments, the first and second protease sites are YES gates independently selected from the group including, but not necessarily limited to: A (LAET↓G, wherein the downward arrow represents the cleavage site), S (LPES↓G, wherein the downward arrow represents the cleavage site), T (ENLYFQ↓S, wherein the downward arrow represents the cleavage site), V (ETVRFQ↓S, wherein the downward arrow represents the cleavage site), and C (LEVLFQ↓GP, wherein the downward arrow represents the cleavage site). YES gate A is proteolytically cleavable by the protease eSrtA(2A9), YES gate S is proteolytically cleavable by the protease eSrtA(4S9), YES gate T is proteolytically cleavable by an evolved potyviral TEV protease, YES gate V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and YES gate C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0075] As disclosed herein, multiple conditional gates can be combined to form layered and higher-order logical structures. Accordingly, in embodiments, the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0076] In example embodiments, a biological circuit comprises a 5-input- responsive [AND / (OR)]OR(AND) gate. While any protease recognition site can be used for each gate, in example embodiments, the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide (i.e., wherein A comprises the polypeptide sequence LAET↓G, S comprises the polypeptide sequence LPES↓G, T comprises the polypeptide sequence ENLYFQ↓S, V comprises the polypeptide sequence ETVRFQ↓S, and C comprises the polypeptide sequence LEVLFQ↓GP; downward arrows within the polypeptide sequences represent cleavage sites). Logic gates A, S, T, V, and C are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus- 3C (HRV-3C) protease, respectively.

[0077] Depending on the logic of the biological circuit embodied by the polypeptide, the polypeptide can be comprised of one or more branched polypeptide3915-P1351WO.UW -17-structures, one or more non-branched polypeptide structures, or any combination thereof, without departing from the scope and spirit of the disclosure. In embodiments, the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof. In embodiments, the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins. In embodiments, the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group including, but not necessarily limited to: SpyLigation, SnoopLigation, and DogLigation. Biomaterials

[0078] The disclosure also provides biomaterials that are configured for conditional release of cargo therefrom, which are biocompatible and can be biodegradable, in at least some embodiments. Biomaterials can comprise one or more polypeptides, as disclosed herein, for logic operations and coordinated release of cargo only under appropriate circumstances. Biomaterials can be implantable into a mammalian or human body for smart monitoring of drug levels, metabolite levels, toxin or poison levels, or the like, for smart release of drug, anti-toxin or antidote, or the like, into the body only as needed.

[0079] Accordingly, in an aspect, the disclosure provides a biomaterial comprising a biological circuit configured for a conditional release of a cargo from an immobilized portion of the biomaterial. The biological circuit comprises a polypeptide comprising a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide. The cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; in the event that one or more protease sites is / are proteolytically cleaved, the conditional release occurs for release of the cargo from the biomaterial.

[0080] In embodiments, the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of3915-P1351WO.UW -18-the biomaterial. In embodiments, the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial. In embodiments, the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0081] In embodiments, the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0082] In embodiments, the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0083] In embodiments, the biological circuit comprises a 5-input-responsive [AND / (OR)]OR(AND) gate. In embodiments, the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

[0084] In embodiments, the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

[0085] In embodiments, the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins. In embodiments, the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group consisting of:3915-P1351WO.UW -19-SpyLigation (i.e., the ligation reaction between SpyCatcher and SpyTag), SnoopLigation (i.e., the ligation reaction between SnoopCatcher and SnoopTag), and DogLigation (i.e., the ligation reaction between DogCatcher and DogTag).

[0086] In embodiments, an immobilized portion of the biomaterial comprises a solid support and the conditional release occurs for release of the cargo from the solid support. In embodiments, a biomaterial comprises a liquid matrix that comprises a biological agent configured to bind to the cargo. In embodiments, a biological agent is a cell or a portion thereof that comprises an antigen, and the cargo is an antibody or a portion thereof that comprises an antigen-binding domain. Implementations

[0087] Aspects and embodiments of the disclosure, including but not necessarily limited to biomaterials and polypeptides, can be implemented into any of a variety of forms for use in a particular application. For example, in various embodiments, a polypeptide of a biomaterial can be encoded by a nucleic acid comprising a polynucleotide sequence encoding the polypeptide. In embodiments, the polynucleotide sequence comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identity with at least one sequence, or at least one portion of a sequence, selected from the group including, but not necessarily limited to: SEQ ID NOs:1- 37. Furthermore, an expression cassette and / or expression vector can comprise a polynucleotide sequence that is configured for expression of a polypeptide in a biological system. Other implementations can include a genetically modified host cell comprising a nucleic acid, a host organism comprising a genetically modified host cell, or the like.

[0088] In various aspects, the disclosure provides a biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising a biomaterial, a genetically modified host cell, or both. In embodiments, an implantable biosensor, implantable medical device, or implantable drug delivery system is biocompatible and can be biodegradable, for example, after substantive or exhaustive release of cargo therefrom, such that surgical removal of the implanted device after use is not necessary. Terminology3915-P1351WO.UW -20-

[0089] Unless stated otherwise, experimental hypotheses or forward-looking models and statements are not intended to be binding on the applicant or exhaustive of the range of possible experimental hypotheses or forward-looking models and statements, but rather are intended to be illustrative, non-limiting examples for aiding those in the art in the understanding and practice of elements of the disclosure.

[0090] As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. Unless otherwise required by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0091] As used herein, the term “biological system” refers to a living or non- living mixture of biological factors. Non-limiting examples of biological systems include cells, cell cultures, cell-free systems, tissues, tissue cultures, organs, organ cultures, and organisms.

[0092] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0093] Unless the context clearly requires otherwise, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claim.

[0094] Unless the context clearly requires otherwise, the phrase “consisting of” excludes any element, step, or ingredient not specified.

[0095] If an element is described or claimed herein such that it “comprises” a feature, that description or claim also includes embodiments wherein the element “consists essentially of” and embodiments wherein the element “consists of” the feature, unless something else is specifically stated to the contrary.

[0096] As used herein, the term “biomaterial” refers to a material that is biocompatible and, optionally, biodegradable. A biomaterial can be compatible with an interior of a mammalian or human body and can be used as a component of an implantable medical device, implantable biosensor, implantable drug delivery device, or the like.

[0097] As used herein, the term “biological circuit” refers to a biochemical construct, for example a polypeptide of the disclosure, that comprises one or more Boolean logic gates as disclosed herein. A biological circuit is configured for the performance of3915-P1351WO.UW -21-logic operations and the conditional release of a cargo from an immobilized portion of a biomaterial.

[0098] As used herein, the term “cargo” refers to a chemical or biochemical that is tethered, trapped, or otherwise immobilized or affixed to an immobilized portion of a biomaterial prior to being released from the biomaterial because of a conditional release. The conditional release is a physical release of the cargo from the biomaterial that occurs due to one or more required conditions being met, as disclosed herein.

[0099] As used herein, the term “portion,” including as used in the phrase “portion of a polypeptide,” refers to a part of the polypeptide that is less than the whole of the polypeptide.

[0100] As used herein, the term “immobilized portion” including as used in the phrase “immobilized portion of a biomaterial” refers to a solid or semi-solid part of the biomaterial that is not mobile in the presence of a fluid or liquid phase. An immobilized portion of a biomaterial can comprise a solid support such that a conditional release results in release of a cargo from the solid support into a liquid matrix (e.g., a liquid matrix that is adjacent to or that surrounds the solid support). Non-limiting examples of solid supports include biocompatible gels or hydrogels, implantable pills or tablets or microcontainers that are configured for conditional release of a cargo therefrom, intravenous tubing configured for conditional release of a cargo therefrom in response to a blood clotting reaction, or the like.

[0101] As used herein, the term “liquid matrix” is open-ended and refers to any liquid that is adjacent to or that surrounds an immobilized portion of a biomaterial. Non- limiting examples of liquid matrices include non-biological liquid matrices including but not limited to water, wastewater, well water, municipal water, food or food products, drink or drink products, food or drink processing chemicals, food or drink ingredients or precursors, or the like, as well as biological liquid matrices including but not limited to any of various bodily fluids including but not limited to blood, extracellular fluid, urine, cerebrospinal fluid, bile, stomach acid, saliva, sweat, tears, stool, and the like.

[0102] As used herein, the term “conditional release” refers to a physical release of a cargo from an immobilized portion of a biomaterial as a result of one or more conditions required for the physical release being met.

[0103] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and3915-P1351WO.UW -22-without limitation, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice, as well as non-mammalian species, such as shark immunoglobulins. The term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have abinding constant for the molecule of interest that is at least 103M−1or greater, at least 104M−1or greater or at least 105M−1or greater than a binding constant for other moleculesin a biological sample). The term “antibody” also includes genetically engineered forms of intact antibodies or antibody fragments such as chimeric antibodies, humanized antibodies (e.g., humanized murine antibodies), multispecific antibodies (whether heteroconjugate or fusion proteins; e.g., bispecific antibodies), and single chain antibodies (e.g., single chain Fvs (scFvs)), diabodies, minibodies, and nanobodies. The term “antibody” is thus used expansively to include any protein that comprises an antigen-binding site and is capable of binding to its antigen.

[0104] In typical variations, an antibody comprises at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. In embodiments, an antibody is composed of both a heavy chain and a lightchain, each of which has a variable region, termed the variable heavy (VH) region and thevariable light (VL) region.

[0105] Typically, an intact immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chain, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined; the Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and3915-P1351WO.UW -23-heavy chains, largely adopt β-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the β-sheet structure. Thus, framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non- covalent interactions.

[0106] The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified bythe chain in which the particular CDR is located. Thus, a VHCDR3 is located in the variabledomain of the heavy chain of the antibody in which it is found, whereas a VLCDR1 is theCDR1 from the variable domain of the light chain of the antibody in which it is found.Antibodies that bind IFN-γ or IL-6 will have specific VHand / or VLregion sequences, andthus specific CDR sequences for the antigen to which the antibody binds. Antibodies with different specificities (i.e., different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are called specificity determining residues (SDRs).

[0107] The term “antibody” is further intended to encompass digestion fragments, specified portions, derivatives, and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and / or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof. Examples of binding fragments encompassed within the term “antigen binding portion” of an antibody include a Fab fragment, a monovalent fragment consistingof the VL, VH, CLand CHdomains; a F(ab′)2 fragment, a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consistingof the VHand CHdomains; a Fv fragment consisting of the VLand VHdomains of a singlearm of an antibody, a dAb fragment, which consists of a VHdomain; and an isolatedcomplementarity determining region (CDR). Furthermore, although the two domains of theFv fragment, VLand VH, are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be made as a single proteinchain in which the VLand VHregions pair to form monovalent molecules (known as singlechain Fv (scFv)). Single chain antibodies are also intended to be encompassed within the term “fragment of an antibody.” Any of the above-noted antibody fragments can be3915-P1351WO.UW -24-obtained using molecular biological techniques, and the fragments can be screened for binding specificity and neutralization activity in the same manner as are intact antibodies.

[0108] “Antibody fragments” or “antigen binding fragments” include proteolytic antibody fragments (such as F(ab′)2 fragments, Fab′ fragments, Fab′-SH fragments, and Fab fragments), recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)′2 fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (“dsFv”), diabodies, and triabodies, and camelid antibodies. An scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.

[0109] As used herein, the term “antibody derivative” is intended to encompass molecules that bind an epitope as defined herein and which are modifications or derivatives of an isolated antibody. Derivatives include, but are not limited to, for example, bispecific, heterospecific, trispecific, tetraspecific, multispecific antibodies, diabodies, chimeric, recombinant, and humanized. As used herein, the term “bispecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities. As used herein, “multispecific molecule” or “heterospecific molecule” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities. As used herein, the term “heteroantibodies” refers to two or more antibodies, antibody binding fragments (e.g., Fab), derivatives thereof, or antigen binding regions linked together, at least two of which have different specificities.

[0110] The term “antibody variant” is intended to include antibodies produced in a species other than a rabbit. It also includes antibodies containing post-translational modifications to the linear polypeptide sequence of the antibody or fragment. It further encompasses fully human antibodies.

[0111] As used herein, the term “antigen” refers to a compound, composition, or substance that can be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, and proteins. Common categories of3915-P1351WO.UW -25-antigens include, but are not limited to, protein-based biological factors, cytokines, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.

[0112] As used herein, “binding affinity” refers to the tendency of one molecule to bind (typically non-covalently) with another molecule, such as the tendency of a member of a specific binding pair for another member of a specific binding pair. A binding affinity can be measured as a binding constant, which binding affinity for a specific binding pair(such as an antibody / antigen pair) can be at least 1×10−5M, at least 1×10−6M, at least1×10−7M, at least 1×10−8M, at least 1×10−9M, at least 1×10−10M, at least 1×10−11Mor at least 1×10−12M. Binding affinity can be calculated by a modification of the Scatchardmethod, an antigen / antibody dissociation rate, or a competition radioimmunoassay. A highbinding affinity for an antibody / antigen pair can be at least about 1×10−8M, at least about1.5×10−8M, at least about 2.0×10−8M, at least about 2.5×10−8M, at least about 3.0×10−8M, at least about 3.5×10−8M, at least about 4.0×10−8M, at least about 4.5×10−8M, or atleast about 5.0×10−8M.

[0113] As used herein, the term “biological agent” is inclusive and refers to an agent that is subjected to, or is a target of, at least one biological function of a cargo.

[0114] As used herein, the term “expression cassette” refers to a component of a vector DNA that includes a gene and a regulatory sequence to be expressed by a transfected cell. As used herein, the term “expression vector” refers to a vector DNA that is configured for delivery into a cell or biological system, or organism, for expression.

[0115] Unless stated otherwise herein, 1-letter abbreviations for nucleic acids are consistent with the nomenclature used in the art (i.e., A, Adenine; T, Thymine; C, Cytosine; G, Guanine; U, Uracil). Unless stated otherwise herein, 1-letter abbreviations for amino acids are consistent with the nomenclature used in the art (i.e., Alanine, A; Arginine, R; Asparagine, N; Aspartic acid, D; Cysteine, C; Glutamic acid, E; Glutamine, Q; Glycine, G; Histidine H; Isoleucine, I; Leucine, L; Lysine, K; Methionine, M; Phenylalanine, F; Proline, P; Serine, S; Threonine, T; Tryptophan, W; Tyrosine, Y; Valine, V).

[0116] Unless stated otherwise herein, the terms “nucleic acid,” “amino acid,” “nucleotide,” and “peptide” are inclusive and open-ended, and do not exclude from their scope any chemically modified or post-translationally modified versions of these structures, and also do not exclude from their scope any nuclear modified versions, for3915-P1351WO.UW -26-example, due to the presence of one or more radioisotopes in one or more of these structures.

[0117] Unless otherwise stated or the context clearly requires otherwise, methods of the disclosure can be performed, in whole or in part, in any order of steps, including steps that are performed subsequently, in parallel, and in combination. In addition, methods can be performed, in whole or in part, by humans optionally assisted by one or more machines such as one or more computational devices or systems (e.g., computer(s)). In at least some instances, methods can be performed by one or more humans with little or no substantive assistance by one or more machines. In at least some other instances, methods can be performed by one or more humans with substantive assistance by one or more machines, and in at least some instances, one or more machines can perform methods autonomously or semi-autonomously.

[0118] Unless otherwise stated or required by the context, an agent (e.g., small molecule inhibitor, protein, therapeutic molecule, etc.) of the disclosure can be provided or formulated as an element of a composition for its use in a method of the disclosure. As known in the art, compositions can be water or oil-based, and can include carriers, salts, lipids, surfactants, pH buffers, and the like to configure the composition to be suitable for its intended purpose (e.g., acceptance for biological, pharmacological, or medical applications).

[0119] A composition of the disclosure can be formulated as a pharmaceutical composition. As used herein, “pharmaceutical composition” refers to a composition that includes at least one pharmacologically active agent in combination with a buffer, a pharmaceutical excipient, a surfactant, a salt, a solvate, a lipid, a phospholipid, a carrier, a diluent, or any combination thereof that preserves and configures the agent for storage, transport, and pharmacological activity.

[0120] As used herein, an “instructional material” includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of one or more elements of a kit of the disclosure for carrying out a method of the disclosure, including methods for biosensing or biomaterial-based alleviation of one or more diseases or disorders as described herein. Optionally, or alternately, the instructional material can describe one or more methods of alleviating diseases or disorders in a cell or a tissue of a mammal. The instructional material of the kit can, for example, be affixed to a container which contains an identified compound or3915-P1351WO.UW -27-biomaterial or can be shipped together with a container which contains the identified compound or biomaterial. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively.

[0121] As used herein, the term “intein” refers to a portion of a polypeptide that is autocatalytically self-spliced from the polypeptide; the term “split intein” refers to an intein that performs protein trans-splicing, wherein two protein fragments bind to form a catalytically competent enzyme, then catalyze their own excision and the ligation of their flanking sequences. As used herein, the term “extein” refers to a portion of a polypeptide that is maintained within the polypeptide as a result of a protease or other enzymatic reaction.

[0122] Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0123] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0124] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0125] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0126] All of the references cited herein are incorporated by reference. Aspects of the disclosure can be modified, if necessary, to employ the systems, functions, and3915-P1351WO.UW -28-concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0127] It will be appreciated that, although specific embodiments of the disclosure have been described herein for purposes of illustration, various modifications can be made without deviating from the spirit and scope of the disclosure. Accordingly, the disclosure is not limited except as stated by the claims. EXAMPLES Example 1. Boolean Logic-gated Protein Presentation via Autonomously Compiled Molecular Topology

[0128] Stimuli-responsive materials enable advanced applications in biosensing, tissue engineering, and therapeutic delivery. Though controlled molecular topology can be an effective route towards creating materials that respond to prespecified input combinations, prior efforts suffer from a reliance on complicated and low-yielding multistep organic syntheses that dramatically limit their utility. Harnessing the power of recombinant expression, this example integrates emerging chemical biology tools to create topologically specified protein cargos that can be site-specifically tethered to, and conditionally released from, biomaterials following user-programmable Boolean logic. Notably, construct topology is autonomously compiled during expression via spontaneous intramolecular ligations, enabling direct and scalable synthesis of advanced operators. Using this framework, this example specifies protein release from biomaterials following all 17 possible YES / OR / AND logic outputs from input combinations of three orthogonal protease actuators, multiplexed delivery of 3 distinct biomacromolecules from hydrogels, 5-input-based conditional cargo liberation, and logically defined protein localization on / within living mammalian cells. Introduction

[0129] Programming stimuli-responsiveness into biomaterials, living cells, and biohybrid construct represents an exciting frontier poised to galvanize many advanced bioengineering applications. For instance, controlled presentation of biomolecules (e.g., proteins, peptides, polysaccharides, nucleic acids) in / from materials holds direct applicability in therapeutic delivery, tissue engineering, organoid development, and the biomanufacturing of value-added chemicals. Co-opting novel methods from organic- and3915-P1351WO.UW -29-synthetic bio-based chemistries, stimuli-responsive material platforms can sense and act upon diverse inputs including light, redox potential, enzyme, and pH. While such approaches can transform biomaterials from static to dynamic systems, most prior platforms exhibit only single-input / single-output-type responses, and this significantly limits their scope of use.

[0130] This disclosure contemplates that next-generation applications, including disease-triggered payload delivery and analyte biosensing, would benefit tremendously from multi-stimuli-responsive, truly “smart” materials that transduce a set of environmental inputs into a conditional, functional output, via a specified Boolean logic. Though logically defined biomolecule release has been accomplished using hydrogel-enzyme hybrids, degradable nanoparticles, and caged self-immolative polymers, prior strategies have been limited in their lack of generalizability, reliance on specific input chemistries, and overall inability to elicit multiplexed operations. As part of development towards imparting more modular Boolean biocomputability into materials, it can be demonstrated that molecular topology can be used to encode advanced environmental responsiveness. In this example, the linker between a biomolecule cargo and a stable material serves as an executable YES gate for programmable release when it contains a single degradable moiety, an OR gate (denoted with logic symbol ∨) when two orthogonal cleavable moieties are included in series, and as an AND gate (denoted by logic symbol ∧) when two orthogonally scissile moieties are present in parallel (FIGs 1A-1C). Importantly, logical gates can be expanded hierarchically to create advanced logical circuits responsive to several stimuli via nested YES / OR / AND operations (FIG.1D). Prior systems have been obtained through multistep organic reactions alongside solid / solution-phase peptide synthesis, however, these were poorly scalable methods whose complexity and low-yielding nature limited their implementation and potential for higher-ordered logical responses. Moreover, these systems cannot be synthesized and / or deployed within cells, precluding many applications for programming living function. Given these caveats, (bio)chemical strategies that permit one-step synthesis of logically releasable cargos, as disclosed herein, enable even higher- ordered logical responses and many applications for programming living function, e.g., in a living biological context.

[0131] Towards expanding logical complexity, improving synthetic accessibility, and exercising operations on / within living cells, it was sought to identify generalizable bio- based methods that enable the direct and facile assembly of topologically specified3915-P1351WO.UW -30-Boolean-responsive cargos. In this regard, recombinant protein expression, augmented with recent advances in chemical biology, were implemented. Recombinant polypeptides are highly monodisperse with a sequence defined by its encoding DNA, able to be produced at scale through well-established fermentation processes, and amenable to the introduction of non-natural functionality via semi-synthesis and / or genetic code expansion. Furthermore, judicious use of engineered motifs permits the creation of heterodox protein architectures; proteins can be cyclized head-to-tail auto-catalytically using split inteins (a method referred to as SICLOPPS, FIG. 1E) or assembled into a variety of bespoke branched structures (e.g., tadpoles, n-armed stars, H-shapes) using Tag / Catcher ligation chemistries (FIG.1F). Though these advances can yield diverse protein topologies starting from simple, singular, plasmid blueprints, such methods have not previously been exploited for creation of stimuli-responsive systems.

[0132] In this example, a modular chemical biology-based framework is introduced to autonomously compile proteins with defined molecular topology that can be site-specifically tethered to, and conditionally released from, biomaterials via nested Boolean operations. Using this template, specification of protein release from biomaterials following all 17 possible YES / OR / AND logic outputs from input combinations of three orthogonal protease actuators is successfully demonstrated, as well as the multiplexed delivery of 3 distinct biomacromolecules from a common hydrogel network via distinct logical operations. This example further highlights the expanded logical complexity enabled via these methods, including unprecedented control over protein liberation using a system that exercises biocomputation following a 5-input Boolean-responsive operator circuit. The disclosed example systems also provide the ability to logically specify protein localization on, and within, living mammalian cells. Autonomous molecular compilation represents a powerful new framework for biosynthesis and offers high specificity over biomolecule presentation in / from materials. Results Autonomous compilation of logic-gated proteins

[0133] Towards establishing design rules that functionally underlie an autonomous molecular compilation, mGreenLantern – a monomeric green fluorescent protein variant recently evolved for enhanced brightness and expression levels – was adapted as a model protein cargo whose extent of release could be readily quantified via its fluorescence. Seeking to utilize cleavable moieties for protein release that are genetically3915-P1351WO.UW -31-encoded, three protease actuators that operate on distinct peptide sequence substrates were encoded: sortase variants eSrtA(2A9) and eSrtA(4S9) which evolved to recognize different pentapeptides (LAET↓G and LPES↓G, respectively referred to as A and S), alongside an evolved potyviral TEV protease (which acts upon sequence ENLYFQ↓S, referred to as T), since this trio of actuators would not have been expected to have exhibited undesirable crosstalk (FIG. 1G). Having identified the mGreenLantern cargo and the three stimuli- labile moieties, it was sought to recombinantly synthesize the 17 distinct logically responsive systems that exhaustively span all possible hierarchical YES / OR / AND combinations. To enable Ni-NTA-based immobilized metal affinity chromatography (IMAC) purification and covalent anchoring onto SpyCatcher-displaying materials, both a 6x-Histidine tag and a SpyTag motif were installed onto each protein regardless of the targeted logical operation.

[0134] With the design criteria established, the three base logical operators were assembled as follows: (1) YES gates (i.e., A, S, T) were designed as constructs, harboring a protease-specific recognition sequence between the mGreenLantern cargo and the SpyTag anchor. (2) OR gates (i.e., A∨S, A∨T, S∨T) followed a similar blueprint, whereby two distinct protease recognition sequences were installed in series between the cargo and material-tethering point. (3) AND gates (i.e., A∧S, A∧T, S∧T) were cyclized head-to-tail using a split Cfa intein via SICLOPPS. To further bias the reaction towards complete splicing and eliminate possible by-products, an SsrA tag was installed at the protein C- terminus to mark all un-spliced (i.e., non-cyclized) product for proteasomal degradation and clearance. Plasmids encoding for the 9 possible YES-, OR-, and AND-gated operators were transformed into BL21(DE3) Escherichia coli. Following expression, proteins were purified by IMAC; cyclic constructs were further purified via size exclusion chromatography (SEC) to separate the target monocyclical constructs from higher-order macrocycle by-products that can result from SICLOPPS (Examples 2-4). In each case, desired proteins were obtained in good yield (YES and OR operators were produced at 30- 50 mg L-1of culture, whereas the AND operators had a final yield of approximately 20 mg L-1of culture), as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and liquid chromatography mass spectrometry (LC-MS) (Example 5). Some higher order macrocycles remained for AND-gated constructs following SEC (i.e., ~15% of the S∧T and A∧T, ~25% for A∧S).3915-P1351WO.UW -32-

[0135] Heartened by the ability to recombinantly produce the base YES-, OR-, and AND-type Boolean gates, each from a singular reading frame, the methods were extended to target higher-order logical operators responsive to 3 distinct inputs: (1) OR / (AND)-gated constructs [i.e., A∨(S∧T), S∨(A∧T), T∨(A∧S)] were designed to undergo macrocyclization through SnoopLigation, whereby installation of the SnoopCatcher motif at an internal site in the protein while keeping the SnoopTag at the N-terminus leads to the creation of a bespoke branched structure which faithfully recreates the tadpole-like geometry useful for the logical operation. Notably, SnoopLigation and SpyLigation are fully orthogonal to each other, permitting complete chemical decoupling of payload configuration and anchoring, analogous to the use of orthogonal synthetic “click” chemistries for material synthesis. (2) AND / (OR)-gated constructs [i.e., A∧(S∨T), S∧(A∨T), T∧(A∨S)] were assembled similarly to the dual-input AND structures, differing in that two of the cleavable sequences were expressed in series to one another and in parallel to the third. (3) The OR / OR-gated construct [i.e., A∨S∨T] harbors the three protease substrate sequences expressed directly in series. (4) The AND / AND construct [i.e., A∧S∧T] relies on two orthogonal Tag / Catcher ligations occurring in tandem to form a bicyclic protein cargo. Here, SnoopTag is installed at the cargo’s N-terminus and a DogTag at the C-terminus, with their concomitant Catchers located internally in the expression frame. Plasmids encoding for these 8 remaining 3-input systems were cloned, transformed into BL21(DE3) cells, expressed, and purified via IMAC / SEC (Examples 2-4). All species were obtained with the desired molecular weights in good yield, as indicated by SDS- PAGE and LC-MS analysis (Example 5). The OR / OR and OR / (AND)-gated species were of high purity; the AND / (OR)-gated constructs had good overall purity with <15% macrocycles present. The A∧S∧T target was correctly identified by whole-protein mass spectrometry, though challenges separating the bicyclic and monocyclic species via SEC limited the final sample purity to ~75%.

[0136] Notably, the 17 distinct logically responsive proteins were autonomously compiled and obtained from singular plasmids via conventional fermentation / purification and characterized over the course of just a few weeks. In direct contrast to prior efforts that were reliant on organic synthesis and / or peptide chemistry, the disclosed example approach features a rapid Design-Build-Test-Learn timeline that enables designs to be quickly sequence-optimized, structurally debugged, and scaled up as needed. Characterization of operator logical response in solution3915-P1351WO.UW -33-

[0137] With the core library in hand, it was next sought to assay the 17 constructs’ in-solution logical response to each of the 8 possible input combinations of eSrt(2A9) (denoted asA), eSrt(4S9) (denoted asS), and TEV (denoted asT) (Example 6) at non- kinetically limited endpoints (Examples 7-9, FIGs 13A-13F, 14A-14F). It was observed that changes in protein mass and / or topology gave rise to altered migration patterns, with full SDS-PAGE gels for all 17 logical operators and 8 treatment conditions presented in FIGs 15A-15G. Through gel densitometry, the extent of response was quantified for each protein to each input actuator set, demonstrating that the genetically encoded logical operators behaved as intended (FIGs 4A-4H). Of note, near-perfect responses were observed with exceptionally high signal / noise ratios for 16 of the 17 operators, and this can be attributed to the simple yet meticulously engineered logical operators and the pristine targeting of orthogonal protease-driven actuation. Though small amounts of macrocycle contaminants were present in some samples, these did not interfere with logical operation or result in off-target release. Owing to monocyclic impurities present in the A∧S∧T construct, gel densitometry analysis was not performed on theAST-releasable cargo. The exhaustive validation of each possible construct demonstrates that complex biocomputation can be achieved with high fidelity through hierarchical nesting of YES / OR / AND logic gates. Characterization of operator logical response from magnetic bead surfaces

[0138] After molecularly validating construct behavior, the focus was shifted towards characterizing the stimuli-responsiveness of the species in a materials’ context. Here, a magnetic bead-based assay was developed, enabling rapid interrogation of each protein’s logical response to input actuator combinations, whereby supernatant fluorescence would indicate protease-driven mGreenLantern release from the solid support. Dibenzocyclooctyne (DBCO)-functionalized magnetic beads were uniformly decorated with a monofunctionalized SpyCatcher-azide protein via strain-promoted azide- alkyne cycloaddition (SPAAC). SpyCatcher-azide was obtained using genetic code expansion, wherein azido-phenylalanine was site-specifically installed via amber suppression near the protein’s C-terminus (Example 10) and served as a linker to immobilize each logic-releasable mGreenLantern protein to the supporting substrate. For each operator type, responsiveness to all eight input combinations involvingA,S, andTwas evaluated (Example 11). Protein release was quantified by measuring supernatant fluorescence at non-kinetically limited endpoints following treatment (FIGs 2A-2B).3915-P1351WO.UW -34-Consistent with the in-solution treatment assays, the signal-to-noise release profiles for 16 of the 17 logical operators were exceptional (FIGs 2C-2I). The A∧S∧T system also yielded the desired response, in that there was significantly higher release accompanying theASTtreatment compared to all other conditions, but with ~25% undesired release in two-inputs (i.e., AS, AT, ST) that was attributed to sample impurity (i.e., the presence of monocyclic 2- input responsive species). These computation signatures represent the most logically advanced protein release from materials to date, likely opening new doors for controlled drug delivery and biosensing. Characterization of operator logical response from hydrogel matrices

[0139] It was next sought to further extend the applicability of this method by programming biomacromolecule release from 3D hydrogel networks, thus assessing the ability to trigger and multiplex signals via the autonomously compiled molecular topology- based approach. To do so, the protein toolkit was first supplemented by expressing and purifying two additional YES-gated fluorescent proteins that are spectrally separated from the startingA-releasable mGreenLantern cargo (mGreenLantern-A, λexc.= 495 nm, λem.= 525 nm). Specifically, an S-releasable mCherry (mCherry-S, λexc.= 580 nm, λem.= 610 nm) and a T-releasable mCerulean (mCerulean-T, λexc.= 433 nm, λem. = 475 nm) were constructed and purified (Example 7) – both also containing a SpyTag motif for material anchoring and 6xHistidine tag for purification. These species responded molecularly to the exhaustive suite of potential inputs, indicated via SDS-PAGE analysis (FIGs 5A-5C, 13A- 13F, 14A-14F). Poly(ethylene glycol) (PEG)-based hydrogels were synthesized through the SPAAC-based step-polymerization of a PEG-tetrabicyclononyne (PEG-tetraBCN, Mn~ 20 kDa) macromer and a triethylene glycol (TEG) diazide in the presence of a small amount of SpyCatcher-azide bound to mGreenLantern-A, mCherry-S, and mCerulean-T via SpyLigation (FIG.3A, Example 12). Such tri-functionalized hydrogels were subjected to each unique input combination involvingA,S, andT, and supernatant blue / green / red fluorescence was quantified to determine each individual protein release (Example 13). Gratifyingly, the three proteins were released independently in the manner specified by their preprogrammed YES-gated input (FIGs 8A-8B).

[0140] Looking to build on this initial success and benefit from the ease of assembly offered by the approach, it was sought to go beyond the multiplexed release of YES operators and layer in different logical operators into the underlying matrix. Toward this end, the SpyCatcher-modified PEG hydrogels were decorated with mGreenLantern-3915-P1351WO.UW -35-A∨T, mCherry-S, and a newly designed and solution-characterized mCerulean-S∧T (FIG. 3B, Example 14, FIGs 5A-5C, 6A-6C). Following treatment with all possible actuator sets, the release of each target biomacromolecule was observed only in response to the appropriate cues or combinations thereof (FIG.3C). Collectively, these studies establish a powerful new route towards the creation of multi-input / multi-output-type materials. Seamless scale-up of logical response and complexity through genetic encodability

[0141] Having successfully demonstrated the promise of autonomously compiled molecular topologies in three different contexts (i.e., in solution, on surfaces, and within hydrogel networks), it was sought to leverage the benefits of biosynthesis to create a 5- input-responsive [AND / (OR)]OR(AND)-gated system, extending well beyond the biocomputational complexity achievable through previous material approaches (FIG.9A). Towards this goal, two additional proteases intended to operate orthogonally to the existing set of inputs were identified: the potyviral Tobacco Vein Mottling Virus (TVMV) protease (operating on substrate ETVRFQ↓S, referred to as V) and the human rhinovirus-3C (HRV- 3C) protease (recognizing LEVLFQ↓GP, denoted as C) (FIG. 9B, Example 6). Having identified the 5 input proteases, it was sought to synthesize the mGreenLantern- [(A∨S)∧T]∨(C∧V) protein, a design which would necessitate the compilation of a genetic blueprint into a bi-cyclic protein (Example 15). To achieve such a logical operation, a recently pioneered “assembly-reaction” synergy approach was adapted, where motifs that assemble through fragment dimerization, catenation, or similar mechanisms bring covalent chemistries (e.g., Tag / Catcher ligations) into close proximity that drive reaction completion. In this example case, it was reasoned that deploying SICLOPPS to cyclize a target protein would bring internal Tag and Catcher motifs into physical proximity, promoting complete ligation to create the pinch point-containing bicyclic protein. Employing this methodology, the mGreenLantern-[(A∨S)∧T]∨(C∧V) was generated solubly, in high purity (>95%) (Example 10) and yield (5 mg L-1of culture following IMAC / SEC), and with the desired molecular mass (FIG.9C).

[0142] To validate the response signature of this logical construct, mGreenLantern-[(A∨S)∧T]∨(C∧V) was immobilized onto SpyCatcher-functionalized magnetic beads (Example 16) and release assessed via supernatant fluorescence following treatment via each of the 32 potential actuator combination sets. The desired anti-interferent penta-input responsiveness was observed, demonstrating protein release only in pre-3915-P1351WO.UW -36-programmed conditions with near-perfect signal-to-noise ratios (FIGs 9D, 17). Excitingly, despite its unprecedented functional complexity, this cargo was generated through recombinant methods and required no extensive debugging / recoding. These studies suggest that the potential for even higher-order logical complexity can be achieved including in view of the number of orthogonally labile peptide motifs and fragment reconstitution chemistries that can be identified. Logic-specified extracellular labeling of mammalian cells

[0143] Having established the ability to logically specify protein presentation on bead surfaces and within hydrogel biomaterials, the focus was shifted towards doing so in the context of living mammalian cells. After validating the cytocompatibility of the various input treatments (Example 17, FIGs 18A-18B), a C∧(A∨T)-gated nanobody – a single- domain antigen-binding protein derived from the heavy-chain of an antibody – was generated against the breast cancer-associated human epidermal growth factor receptor type 2 (HER2) (FIG.5A, Example 18). Here, the HER2 nanobody (HER2Nb) was fused to SpyTag via the cyclic C∧(A∨T) linkage and subsequently conjugated to a SpyCatcher- modified enhanced green fluorescent protein (eGFP). The resulting fusion construct [HER2Nb-C∧(A∨T)-eGFP] was obtained in good purity and intended molecular weight (Example 19, FIG.20), responded as intended following in-solution treatment with the 8 possible input combinations ofA,C, andT(FIGs 21A-21B), and selectively bound to the extracellular membrane of HER2-expressing SK-BR-3 cells (FIG.22).

[0144] Following the treatment of HER2Nb-C∧(A∨T)-eGFP with each of the 8 relevant input combinations, samples were individually incubated with SK-BR-3 cells (Example 19). After nuclear labeling with Hoechst 33342 and confocal imaging, fluorescent labeling of cells yielded results that were consistent with the AND / (OR)- specified logical operation; eGFP signal was not observed for cells treated with AC, CT, orACT, though all other conditions showcased extracellular labeling (FIG. 11B). These findings were corroborated using flow cytometry, calculating the percentage of eGFP positive cells for each protease condition (Example 19, FIG.23). Cells receiving the N, A,C, T, and AT treatments were fluorescently labelled (>97% eGFP+), while those in whicheGFP had been proteolytically cleaved from HER2Nb (i.e., AC, CT, or ACT treatments) were >97% eGFP- (FIG. 11C). Excitingly, these studies demonstrate that more advanced bioactive cargos can be easily incorporated during autonomous molecular compilation, and that resulting logic-based operations can be performed in the presence of living cells.3915-P1351WO.UW -37-Logic-specified localization of proteins autonomously compiled within mammalian cells

[0145] Encouraged by these successes in logically specifying protein binding to extracellular membrane components, it was next sought to examine whether these approaches can be used to both autonomously compile and subsequently execute nested Boolean operations within living mammalian cells. In this regard, it was sought to logically specify whether an expressed protein would translocate to the interior cell membrane or throughout the cytosol. Taking advantage of the system’s genetic encodability, a stable HEK293 cell line was generated via lentivirus transduction that expressed a C∨(T∧V) motif bridging mGreenLantern and a membrane-binding CAAX motif via an OR / (AND)-gated topology (FIG.12A, Examples 20-21); if the correct protease combinations were present, the mGreenLantern cargo would be proteolytically cleaved from the CAAX tag, shuffling it from the membrane to the cytosol. These stably modified cells were subsequently transfected with plasmids that individually encoded for each of the 8 input combinations (i.e.,N,C,T,V,CT,CV,CTV), where each protease was separated by a self-cleaving P2A peptide sequence, and a C-terminal mCherry reporter indicating successful transfection and full plasmid read-through. Protease-expressing cells were fluorescently imaged via confocal microscopy, with cytosolic mGreenLantern largely confined to the designed input combinations (i.e.,C,CT,CV,TV,CTV) (FIG. 12B). Radial distribution of mGreenLantern was calculated using CellProfiler (25 total bins) throughout individual mCherry+cells from each of the 8 treatment conditions. Taking the “cell membrane” as the outermost 20% of these radial bins, the relative amounts of cytosolic mGreenLantern protein was quantified throughout the cell (FIG. 12C-12D, Example 20). As programmed, the mGreenLantern remained membrane-bound via the intact CAAX motif for N, T, and V conditions. Notably, some degree of mGreenLantern presented within the cytosol for the T treatment, which was attributed to proteolytic cleavage of the T linkage prior to SnoopLigation-mediated AND gate formation, thereby removing the CAAX tag from the mGreenLantern protein in these cases; this particular result is not necessarily unexpected, given that the proteases and C∨(T∧V) construct are being co-expressed within the same cell. These studies highlight the ability to both autonomously compile and logically dictate protein presentation within mammalian cells, enabling a wide variety of cell and tissue engineering applications. Discussion3915-P1351WO.UW -38-

[0146] In this example, autonomous macromolecular compilation is harnessed to create bioactive proteins that can be site-specifically tethered to, and conditionally released from, biomaterials and cells following user-programmable and hierarchically nested Boolean YES / OR / AND molecular operations. This example demonstrates the modularity and scalability of recombinant expression, multiplexed and multi-stimuli-triggered protein release from biomaterials, including release through a 5-input-responsive [AND / (OR)]OR(AND)-gated system, as well as the logically defined protein localization on / within living mammalian cells. The example design rules yield species that are structurally defined, yet functionally complex, all through direct and unsupervised biosynthesis.

[0147] The power of the presented approach lies in its modularity. Though this example utilized 5 orthogonal proteases to serve as inputs, polypeptide motifs that sense and respond to other input types (e.g., light, small molecule) can be readily implemented to afford additionally engineered programmability. Moreover, alternative approaches that map non-proteinaceous inputs onto protease function and / or expression can be readily implemented; protease bioactive can be conditionally regulated with alternative inputs (e.g., small molecule-mediated heterodimerization of split protein pairs, direct photoactivation), just as protease expression can be selectively induced (e.g., heat shock promotors, optogenetic activators, small molecule addition, CRISPRa).

[0148] Beyond the fluorescent cargos initially adopted for their ease of characterization, Boolean-actuated release of more exotic bioactive proteins (e.g., enzymes, growth factors) can be attained. While this example utilized SpyTag / Catcher ligation to tether proteins to materials, this chemistry can be altered for alternative (non)covalent reactions, including those involving de novo designed heterodimer interactions or click-type handle installation via genetic code expansion, without departing from the scope and spirit of the disclosure. Finally, as the systems are fully genetically encoded, additional opportunities undoubtedly exist to both create and functionally deploy these platforms in cellulo, as well as in vivo. Owing to its powerful design and plug-and- play potential, the disclosed approach enables a host of applications in areas including but not limited to highly targeted drug delivery, on-demand biosensor engineering, and next- generation protein architecture development and refinement. Example 2. General method for protein expression3915-P1351WO.UW -39-

[0149] BL21(DE3) Escherichia coli cells were transformed with the proper operator plasmid construct and grown at 37 °C with 220 rpm agitation to an optical density (OD) of 0.6-0.8 in lysogeny broth (LB) supplemented with kanamycin. Isopropyl β-D-1- thiogalactopyranoside (IPTG) was used to induce protein expression (0.5 mM final concentration for linear constructs, 0.4 mM for cyclic constructs through SICLOPPS or SnoopLigation), at which point the cultures were moved to a reduced temperature (18 °C) for 18 hours. Cells were then harvested via centrifugation (4,000g, 20 min, 4 °C) and either (1) flash-frozen and stored at -80 °C, or (2) directly processed for purification. Example 3. Purification of protein constructs via IMAC and SEC Immobilized metal affinity chromatography (IMAC)

[0150] Cell pellets were reconstituted in lysis buffer (40 mL, 20 mM Tris, 50 mM NaCl, pH = 7.5). These were then supplemented with phenylmethylsulfonyl fluoride (PMSF, 1 mM final concentration) and sonicated on ice (18 min at 30% amplitude and 33% duty cycle) (Fisher Scientific, Waltham, MA). After sonication, lysates were centrifuged (45 min, 11,000g) to separate soluble from insoluble fractions.

[0151] Clarified lysates were loaded onto an ÄKTA Pure 25L FPLC (Cytiva, Marlborough, MA) equipped with a HisTrap HP column (5mL) at a flow rate of 5 mL min-1. The HisTrap column was first equilibrated with 5 column volumes of lysis buffer prior to loading. Once all protein was bound, the column was washed with >10 column volumes of lysis buffer until residual UV read-outs (λ = 280 nm) dropped to 0 absorbance units (au). Target protein was then eluted off the column into a 96-well plate by switching to elution buffer (lysis buffer + 250 mM imidazole). Eluate fractions were preliminarily assessed for purity via sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE); the purest fractions were then pooled pending further dialysis for size-exclusion chromatography (SEC). Protein yield was determined by measuring pooled elution concentration (NanoDrop, A280 absorbance), then back calculating final yield per liter of culture.

[0152] For linear constructs that had no higher order macro-cycle contaminants, the pooled purified protein was dialyzed (20 mM Tris, 50 mM NaCl, pH = 7.5) to remove imidazole (SnakeSkin™ Dialysis Tubing, 10K MWCO, Fischer Scientific, Waltham, MA). After >4 dialysis bath changes, proteins were spin-concentrated if needed using an Amicon Ultra 15 centrifugation filter (10 kDa MWCO, Sigma-Alrich, Burlington, MA). The3915-P1351WO.UW -40-purified solution was supplemented with glycerol (10% of final volume) to act as cryoprotectant, aliquoted into single-use tubes to avoid repeated freeze-thaw cycles, and flash-frozen with liquid nitrogen prior to long-term storage. Size exclusion chromatography (SEC)

[0153] For cyclized constructs, IMAC-purified proteins were loaded onto an ÄKTA Pure unit equipped with a HiLoad 26 / 600 Superdex 75pg SEC column. The column was pre-equilibrated with a high salt buffer solution (20 mM Tris, 300 mM NaCl, pH = 7.5). Sample injection occurred at a flowrate of 2.6 mL min-1. Buffer flowrate through the system occurred at 1.5-1.8 mL min-1, depending on the purity of the injected solution. Purified eluates were collected in 96-well tubes. Prior to pooling, preliminary SDS-PAGE was conducted to verify fraction purity. Gel densitometry was performed to determine final sample purity and presence of macrocycles. The total lane intensity was determined, then the intensity of impurities (e.g., macrocycles) was measured. The percentage of impurities could then be calculated. All products were considered pure unless otherwise reported in the text. Proteins were then spin-concentrated using an Amicon Ultra 15 centrifugation filter (10 kDa MWCO, Sigma-Alrich, Burlington, MA). The purified solution was supplemented with glycerol (10% of final volume) to act as cryoprotectant, aliquoted into single-use tubes to avoid repeated freeze-thaw cycles, and flash-frozen with liquid nitrogen prior to long-term storage. Example 4. Plasmid construction for Boolean-releasable mGreenLantern species

[0154] Cloned plasmids [pET-29b(+), with desired sequences inserted between NdeI and XhoI cloning sites] were ordered from GenScript (Piscataway, NJ). The open reading frames (ORFs) of the 17 YES / OR / AND nested logical operators are given below (5’ 3’).2A9c, 4S9c, and TEVc respectively denote the recognition sequences for their associated enzymes. All mGreenLantern constructs were designed to contain a SpyTag for material tethering and a 6xHis motif for IMAC-assisted purification. mGreenLantern-A MGSS-mGreenLantern-GGS(x3)-2A9c-GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGGAGTTCTATGGTATCAAAAGGAGAAGAGTTGTTTACCGGTGTTGTTCCGATTCTGGTCGAG TTAGATGGCGATGTTAACGGCCACAAGTTCAGCGTTCGTGGTGAGGGTGAAGGCGACGCCACCAATGGTAAA CTGACCTTGAAGTTCATCTGCACCACGGGTAAGCTGCCTGTTCCGTGGCCGACCCTGGTAACTACGCTGGGT3915-P1351WO.UW -41-TATGGTGTTGCATGTTTTGCGCGTTACCCGGACCACATGAAACAACATGATTTCTTTAAGTCTGCAATGCCG GAAGGTTACGTCCAAGAAAGAACAATTAGCTTTAAGGACGACGGTACTTACAAAACCCGCGCGGAGGTGAAA TTCGAGGGCGACACCCTGGTGAACCGTATTGTGCTGAAAGGCATCGACTTCAAAGAGGACGGCAACATCCTG GGTCATAAGCTGGAATATAACTTCAACAGCCACAAAGTTTATATCACCGCCGACAAACAAAAAAACGGGATC AAAGCTAATTTTAAGACCCGTCATAACGTGGAGGATGGCGGCGTTCAGCTGGCTGACCACTACCAGCAGAAC ACCCCGATTGGTGACGGTCCGGTCTTGCTGCCAGATAATCATTACTTGAGCCATCAGAGCAAGTTGAGCAAA GATCCGAATGAAAAACGCGATCACATGGTGCTTAAGGAGCGCGTGACGGCGGCGGGTATTACCCATGACATG GATGAACTGTATAAGGGCGGCAGCGGCGGCTCCGGCGGCTCTCTGGCGGAAACGGGTGGTGGAAGCGGCGGT TCGGGTGGTAGTCGTGGTGTGCCGCATATCGTGATGGTTGATGCGTATAAGCGTTACAAGGGTGGGTCCGGT GGCTCCGGCGGCTCGCACCACCACCACCACCAC (SEQ ID NO:1) mGreenLantern-S MGSS-mGreenLantern-GGS(x3)-4S9c-GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGGTCATCTATGGTAAGTAAAGGAGAAGAGCTGTTTACCGGTGTCGTGCCGATTCTGGTTGAA CTGGATGGCGACGTGAACGGTCATAAGTTCAGCGTTCGTGGTGAAGGCGAGGGTGACGCCACCAACGGTAAG TTGACCTTGAAGTTCATCTGCACCACGGGTAAGCTTCCGGTTCCGTGGCCGACCTTGGTGACGACCCTGGGT TATGGTGTCGCGTGTTTTGCCCGTTACCCGGACCACATGAAACAGCATGACTTCTTTAAAAGCGCAATGCCG GAAGGCTACGTTCAAGAGCGCACCATTAGCTTTAAGGACGACGGAACCTACAAGACGAGAGCGGAAGTGAAA TTCGAGGGTGACACCCTGGTCAACCGTATTGTGCTGAAAGGCATCGATTTCAAAGAGGATGGTAATATTCTG GGCCATAAATTGGAATATAACTTTAACAGCCACAAGGTGTATATCACGGCTGATAAACAAAAAAACGGCATC AAAGCTAATTTCAAGACTCGTCATAATGTTGAGGACGGCGGCGTGCAACTGGCAGACCACTACCAGCAGAAT ACCCCGATCGGCGACGGCCCTGTTCTGCTGCCAGACAACCACTACCTGAGCCATCAGAGCAAACTCTCGAAA GATCCGAATGAAAAACGTGATCACATGGTTTTAAAAGAACGCGTGACTGCGGCGGGTATCACCCATGATATG GATGAGCTGTATAAGGGTGGCTCCGGCGGTTCTGGCGGTTCCCTGCCGGAGAGCGGTGGGGGCTCCGGCGGT TCTGGTGGTAGCCGTGGCGTACCGCATATTGTTATGGTTGATGCGTATAAGCGCTACAAGGGCGGTAGTGGC GGTAGCGGTGGTTCTCACCACCACCACCACCAC (SEQ ID NO:2) mGreenLantern-T MGSS-mGreenLantern-GGS(x3)-TEVc-GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGGTCATCTATGGTAAGTAAAGGAGAAGAGTTGTTTACCGGTGTCGTGCCCATCCTGGTCGAG TTAGACGGCGACGTTAACGGTCATAAGTTCAGCGTTCGTGGCGAGGGTGAAGGTGACGCCACCAACGGCAAA CTGACCTTGAAGTTCATCTGCACCACGGGTAAATTACCGGTCCCGTGGCCGACCTTGGTGACGACCCTTGGT TACGGCGTGGCGTGTTTTGCCCGTTACCCGGACCACATGAAACAGCATGACTTCTTCAAAAGCGCAATGCCG3915-P1351WO.UW -42-GAAGGTTACGTTCAAGAGCGCACCATTAGCTTTAAGGATGATGGCACCTATAAAACCCGTGCGGAAGTTAAA TTCGAGGGCGACACCCTGGTGAACCGTATTGTGCTGAAGGGCATCGATTTCAAGGAGGACGGAAACATTCTC GGTCACAAGCTGGAATATAACTTCAACAGCCACAAGGTGTATATCACTGCGGACAAACAAAAAAATGGCATC AAGGCTAATTTTAAGACTCGCCATAATGTTGAGGACGGTGGTGTGCAACTGGCTGACCATTACCAGCAGAAC ACCCCGATTGGTGACGGTCCGGTTTTGCTGCCAGATAATCACTACCTGAGCCACCAGAGCAAGCTGAGCAAA GATCCGAATGAAAAAAGAGATCACATGGTTCTGAAAGAGCGTGTTACGGCGGCAGGCATCACCCATGATATG GATGAACTGTACAAGGGCGGCAGCGGCGGCTCGGGTGGTTCGGAAAACCTGTATTTTCAGTCAGGTGGCTCC GGTGGTTCTGGCGGTTCTCGTGGTGTTCCGCATATTGTGATGGTAGATGCGTATAAGCGCTACAAGGGCGGC TCCGGTGGCTCCGGTGGCTCTCACCACCACCATCACCAC (SEQ ID NO:3) mGreenLantern-A∨S MGSS-mGreenLantern-GGS(x3)-2A9c-GGS(x3)-4S9c-GGS(x3)-SpyTag003- GGS(x3)-6xHis ATGGGGTCAAGTATGGTAAGCAAAGGAGAAGAGTTGTTTACCGGTGTGGTGCCGATTCTGGTTGAA CTGGATGGCGACGTTAACGGTCACAAGTTCAGCGTGCGTGGTGAGGGTGAGGGCGACGCTACCAATGGTAAA TTGACTTTGAAGTTCATCTGTACCACTGGTAAACTGCCGGTTCCGTGGCCTACGTTGGTTACCACGCTGGGT TATGGTGTAGCCTGCTTCGCCCGTTACCCGGATCACATGAAACAACACGACTTCTTCAAAAGCGCAATGCCG GAAGGTTACGTGCAAGAACGTACCATTTCTTTTAAGGACGACGGCACCTACAAAACCAGAGCGGAGGTTAAA TTTGAAGGCGACACCTTGGTCAACCGTATCGTGCTCAAGGGTATTGATTTTAAAGAGGATGGTAATATTCTG GGTCATAAGCTGGAATATAACTTTAACAGCCACAAGGTGTATATCACTGCGGACAAACAGAAAAACGGCATC AAAGCAAATTTCAAGACCCGTCATAACGTGGAGGACGGTGGCGTGCAGCTGGCGGATCACTACCAGCAAAAC ACCCCGATCGGCGACGGTCCGGTTCTGCTGCCGGATAACCATTACCTGAGCCATCAGAGCAAACTGAGCAAA GATCCGAATGAAAAACGCGACCACATGGTTCTTAAGGAGCGCGTGACCGCGGCGGGTATCACGCATGATATG GATGAACTGTATAAGGGTGGTTCCGGCGGCAGCGGCGGAAGCCTGGCTGAAACCGGTGGCGGCAGCGGTGGG TCCGGCGGCTCGTTACCGGAGTCAGGCGGCGGCTCCGGCGGCAGCGGTGGTTCTCGTGGTGTCCCACATATT GTTATGGTTGATGCGTATAAGCGCTACAAGGGTGGCTCCGGAGGTAGTGGTGGTTCTCACCATCACCACCAC CAC (SEQ ID NO:4) mGreenLantern-A∨T MGSS-mGreenLantern-GGS(x3)-2A9c-GGS(x3)-TEVc-GGS(x3)-SpyTag003- GGS(x3)-6xHis ATGGGGAGTTCTATGGTATCAAAAGGAGAAGAGCTGTTTACCGGTGTTGTTCCAATCTTGGTCGAG CTGGATGGTGATGTAAATGGCCACAAGTTCAGCGTTCGTGGTGAAGGTGAAGGTGACGCGACCAACGGTAAA3915-P1351WO.UW -43-TTGACCCTGAAGTTCATCTGCACCACGGGCAAACTGCCTGTTCCGTGGCCGACCCTCGTGACCACGCTTGGC TACGGCGTGGCATGTTTTGCCCGTTACCCGGACCACATGAAACAGCATGACTTCTTTAAAAGCGCAATGCCG GAAGGCTACGTTCAGGAGAGAACAATTAGCTTTAAGGATGATGGCACCTATAAGACCCGTGCGGAAGTTAAA TTCGAGGGCGACACCTTGGTGAACCGTATTGTGTTAAAGGGCATCGATTTCAAAGAGGACGGTAATATTCTG GGTCATAAGTTGGAATATAACTTTAACAGCCATAAGGTGTATATCACTGCCGATAAACAAAAAAACGGCATC AAAGCTAATTTCAAGACGCGTCATAACGTTGAGGACGGTGGTGTGCAACTGGCGGACCACTACCAGCAAAAC ACCCCGATTGGTGACGGTCCGGTTCTGCTGCCGGATAATCATTACCTGAGCCACCAGAGCAAACTTAGCAAG GACCCGAATGAAAAACGCGATCACATGGTCCTGAAAGAGCGCGTGACTGCGGCGGGTATCACCCATGACATG GACGAACTGTATAAGGGCGGTAGTGGCGGTTCTGGTGGTTCCCTGGCTGAGACGGGTGGTGGCTCCGGCGGT TCCGGCGGTTCGGAAAACCTGTACTTCCAGAGCGGGGGTAGCGGTGGCTCGGGGGGCTCTCGTGGTGTGCCG CATATTGTAATGGTCGATGCGTATAAGCGCTACAAGGGTGGATCCGGCGGTTCTGGTGGCTCTCACCACCAC CACCACCAC (SEQ ID NO:5) mGreenLantern-S∨T MGSS-mGreenLantern-GGS(x3)-TEVc-GGS(x3)-4S9c-GGS(x3)-SpyTag003- GGS(x3)-6xHis ATGGGGTCAAGTATGGTAAGCAAAGGAGAAGAGCTGTTTACCGGTGTTGTCCCGATTCTGGTCGAA CTGGATGGCGACGTGAATGGTCATAAGTTCTCCGTGCGTGGTGAGGGCGAGGGTGACGCGACCAATGGCAAA TTGACCCTTAAGTTCATCTGCACCACGGGTAAACTGCCAGTTCCGTGGCCTACATTGGTGACCACCCTGGGT TATGGCGTTGCGTGTTTTGCCCGTTACCCGGACCACATGAAACAGCACGACTTTTTCAAGAGCGCAATGCCG GAGGGTTATGTTCAAGAACGCACCATTAGCTTTAAGGATGACGGCACCTATAAGACGCGTGCTGAAGTTAAA TTCGAAGGCGACACCTTGGTCAACCGTATTGTTCTGAAAGGGATCGATTTCAAGGAGGATGGTAACATTCTT GGTCACAAATTGGAGTACAACTTCAACAGCCACAAGGTGTATATCACGGCTGATAAACAAAAAAACGGTATT AAAGCCAATTTTAAGACCCGTCATAACGTGGAGGACGGTGGCGTGCAGCTGGCAGACCATTACCAGCAAAAC ACCCCGATCGGCGACGGTCCGGTTCTGCTGCCGGATAATCATTACCTGTCCCATCAGAGCAAACTCAGCAAG GATCCGAATGAAAAAAGAGATCACATGGTTCTGAAGGAGCGCGTGACTGCGGCGGGTATCACGCATGATATG GATGAATTATACAAAGGTGGCTCTGGTGGCTCAGGCGGCAGCGAAAACCTGTACTTCCAGAGCGGTGGTAGC GGCGGCTCTGGAGGCTCGCTGCCGGAAAGCGGCGGTGGTTCCGGCGGATCCGGCGGTAGCCGTGGTGTGCCG CATATCGTAATGGTGGACGCGTATAAGCGCTACAAGGGTGGTTCTGGAGGCTCGGGTGGTAGCCACCACCAC CACCACCAC (SEQ ID NO:6) mGreenLantern-A∧S MGSS-CfaC-GGS(x3)-2A9c-GGS(x3)-mGreenLantern-GGS(x3)-4S9c-GGS(x3)- SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA3915-P1351WO.UW -44-ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAGTCCCTGGGCACCCAAAACGTTTATGACATCGGC GTCGAGAAAGACCACAACTTCCTGCTGAAGAATGGTCTCGTGGCGAGCAACTGCTTTAACGGTGGCTCCGGT GGATCGGGTGGTAGCCTGGCGGAGACGGGTGGTGGTTCTGGCGGTTCGGGGGGCTCAATGGTAAGTAAGGGC GAGGAACTGTTTACCGGAGTCGTGCCGATCTTGGTCGAGCTGGACGGCGATGTGAACGGCCATAAGTTCAGC GTTCGTGGTGAAGGTGAGGGCGACGCTACCAATGGTAAGCTCACCTTGAAGTTCATCTGCACCACGGGTAAA TTGCCAGTGCCGTGGCCTACCCTGGTGACCACCTTAGGCTACGGTGTTGCGTGTTTCGCGCGTTATCCGGAC CACATGAAACAACATGACTTCTTCAAGAGCGCCATGCCGGAAGGCTATGTTCAAGAGAGAACAATTAGCTTT AAGGACGACGGCACGTATAAAACCCGTGCGGAAGTTAAATTCGAGGGTGACACGTTGGTTAATCGTATCGTG CTGAAGGGTATTGATTTTAAAGAGGATGGAAACATCCTGGGTCATAAACTGGAGTACAATTTCAACAGCCAT AAGGTGTACATTACGGCCGATAAACAAAAAAACGGCATTAAAGCCAACTTCAAGACCCGTCACAATGTTGAA GACGGCGGTGTGCAACTGGCCGATCACTACCAGCAGAACACCCCGATCGGCGACGGTCCGGTTTTGCTGCCG GATAATCATTACCTGAGCCACCAGAGCAAATTAAGCAAGGACCCGAATGAAAAACGTGATCACATGGTTCTC AAAGAGCGCGTGACTGCGGCGGGTATCACCCATGACATGGATGAGCTTTACAAAGGTGGTTCGGGGGGCAGC GGTGGTAGCCTGCCAGAAAGCGGCGGCGGTAGCGGCGGTTCTGGAGGGAGCCGTGGTGTTCCGCATATCGTG ATGGTGGACGCTTACAAGCGCTATAAAGGTGGCTCCGGTGGTTCAGGTGGTTCTCACCACCACCACCACCAC GGCGGTTCTGGCGGCTCCGGCGGTAGCGCTGAGTATTGCTTGTCCTATGACACCGAAATTCTGACCGTGGAG TACGGTTTTCTGCCGATTGGTAAGATCGTTGAGGAACGCATTGAATGTACGGTGTACACTGTCGACAAAAAC GGTTTTGTTTACACCCAGCCGATTGCGCAGTGGCATAATCGTGGTGAACAGGAGGTCTTTGAATACTGCCTG GAAGATGGCAGCATTATCCGCGCAACCAAAGACCACAAATTCATGACGACCGATGGTCAGATGCTGCCGATC GATGAGATCTTTGAACGTGGTCTGGATCTGAAGCAGGTTGATGGCTTGCCGGCAGCGAACGATGAAAACTAT GCACTGGCTGCG (SEQ ID NO:7) mGreenLantern-A∧T MGSS-CfaC-GGS(x3)-2A9c-GGS(x3)-mGreenLantern-GGS(x3)-TEVc- GGS(x3)-SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAAAGCCTGGGCACCCAGAACGTCTACGACATCGGT GTGGAAAAAGACCACAACTTCCTGCTCAAGAACGGCTTGGTAGCCAGCAATTGTTTTAATGGTGGTAGCGGT GGTTCGGGTGGCTCCCTGGCGGAAACCGGCGGTGGTTCTGGTGGCAGCGGCGGAAGCATGGTCAGCAAGGGT GAGGAACTCTTCACCGGTGTGGTTCCGATCCTGGTCGAGCTGGACGGCGACGTTAACGGCCACAAGTTCAGC GTTCGTGGTGAGGGCGAAGGTGACGCCACCAATGGTAAGTTGACCCTAAAGTTCATCTGTACCACGGGAAAG CTGCCGGTGCCGTGGCCTACTTTGGTTACCACCCTGGGGTACGGCGTGGCGTGCTTTGCACGTTACCCGGAT CACATGAAACAACACGATTTTTTTAAAAGCGCTATGCCGGAAGGTTACGTGCAAGAGCGTACCATCAGCTTT3915-P1351WO.UW -45-AAGGATGATGGCACCTATAAAACCCGTGCCGAAGTGAAATTCGAGGGCGACACCCTGGTTAATCGTATCGTG TTAAAGGGCATCGATTTCAAGGAGGACGGTAATATTCTGGGTCATAAATTGGAGTATAACTTCAACAGCCAT AAAGTTTACATTACCGCAGATAAACAGAAAAACGGCATCAAAGCTAATTTCAAGACTCGCCATAACGTGGAG GACGGTGGTGTCCAACTGGCGGATCACTACCAGCAAAACACCCCGATCGGCGATGGACCGGTTCTTCTGCCG GACAACCATTATCTGAGTCACCAGTCGAAGTTGAGCAAGGATCCGAATGAAAAGCGCGATCACATGGTGCTG AAAGAACGTGTTACGGCTGCTGGTATTACACATGATATGGATGAGTTGTACAAGGGTGGCTCCGGCGGTTCT GGTGGTTCGGAAAATCTGTACTTCCAGAGCGGGGGCTCTGGCGGGTCCGGTGGCAGCCGTGGTGTGCCGCAT ATTGTGATGGTGGACGCGTATAAACGTTATAAGGGTGGCTCTGGCGGATCCGGTGGTTCCCATCACCATCAC CACCACGGCGGCTCCGGCGGCTCTGGTGGTTCTGCGGAGTACTGCCTGAGCTATGACACGGAAATTCTGACG GTAGAGTATGGTTTTCTGCCGATTGGCAAGATCGTTGAGGAACGCATCGAGTGCACGGTTTACACCGTGGAC AAGAACGGCTTTGTTTATACGCAACCGATTGCGCAGTGGCATAACCGCGGTGAACAAGAAGTTTTTGAATAC TGCTTGGAGGACGGTAGCATTATTCGTGCAACTAAAGACCACAAATTCATGACCACCGATGGTCAGATGCTG CCAATCGACGAAATCTTTGAAAGAGGTCTGGACCTGAAACAGGTTGATGGCCTGCCAGCAGCGAACGATGAG AACTATGCGCTGGCGGCG (SEQ ID NO:8) mGreenLantern-S∧T MGSS-CfaC-GGS(x3)-TEVc-GGS(x3)-mGreenLantern-GGS(x3)-4S9c- GGS(x3)-SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGATCAAGTGTAAAAATTATATCTAGGAAGAGCCTAGGTACCCAAAACGTTTACGATATTGGT GTGGAAAAAGACCACAACTTTCTGCTTAAGAACGGCCTGGTCGCGAGCAATTGCTTTAATGGAGGTTCTGGT GGTAGTGGTGGCTCGGAAAATCTGTACTTCCAGAGCGGCGGCTCCGGCGGTAGCGGCGGTAGCATGGTCAGC AAGGGTGAAGAATTGTTTACCGGTGTTGTACCAATCTTGGTCGAACTCGATGGTGATGTTAACGGTCACAAG TTCTCTGTTCGTGGTGAGGGCGAAGGCGATGCGACTAACGGTAAGCTGACCTTGAAGTTCATCTGCACGACT GGTAAATTACCGGTTCCATGGCCGACCCTGGTGACCACGCTGGGCTACGGCGTGGCCTGTTTCGCCCGTTAT CCGGATCACATGAAACAGCATGACTTTTTTAAATCCGCTATGCCGGAGGGCTACGTGCAAGAGCGCACCATT AGCTTTAAGGACGACGGCACCTATAAGACCCGTGCTGAAGTGAAATTCGAGGGCGACACCCTGGTGAACCGC ATCGTTTTGAAGGGCATCGACTTCAAAGAAGACGGTAATATCCTGGGTCACAAACTCGAATACAACTTCAAC AGCCATAAAGTCTATATTACCGCAGATAAACAAAAAAACGGCATCAAAGCGAACTTCAAGACCCGCCATAAC GTTGAGGATGGCGGTGTGCAGCTGGCGGATCACTACCAGCAAAACACCCCGATCGGTGACGGTCCGGTTCTG TTGCCGGACAATCATTACCTGAGCCACCAAAGCAAGTTAAGCAAAGACCCGAATGAAAAGCGTGACCACATG GTTCTTAAAGAAAGAGTGACGGCTGCGGGTATTACCCATGATATGGATGAGCTGTATAAAGGTGGGTCGGGG GGGTCCGGTGGCTCCCTGCCGGAGAGCGGTGGTGGCTCTGGCGGTTCTGGAGGTAGCCGTGGTGTTCCGCAT ATCGTGATGGTGGATGCCTACAAGCGTTACAAAGGTGGCAGTGGCGGTTCTGGCGGTTCGCACCATCACCAC3915-P1351WO.UW -46-CATCACGGCGGTAGCGGTGGTAGCGGTGGTTCAGCGGAATATTGTCTGTCCTATGACACGGAGATCTTGACC GTTGAGTATGGGTTTCTGCCGATTGGCAAGATCGTCGAGGAGCGTATCGAATGCACCGTGTACACTGTAGAC AAGAACGGCTTCGTGTATACGCAACCGATCGCGCAGTGGCATAATCGTGGCGAGCAGGAGGTGTTTGAGTAC TGCCTGGAAGATGGCAGCATTATTCGTGCAACGAAAGACCACAAATTCATGACCACCGATGGGCAGATGCTG CCTATTGATGAGATTTTCGAACGCGGCTTAGACCTGAAGCAGGTTGACGGCTTGCCGGCAGCGAATGATGAG AACTATGCACTGGCTGCG (SEQ ID NO:9) mGreenLantern- A∧(S∨T) MGSS-CfaC-GGS(x3)-4S9c-GGS(x3)-TEVc-GGS(x3)-mGreenLantern- GGS(x3)-2A9c-GGS(x3)- SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATATCTAGGAAGAGCCTCGGTACACAAAACGTGTACGACATCGGT GTGGAGAAAGACCACAACTTCCTGCTGAAAAACGGCCTGGTCGCCAGCAACTGCTTCAACGGTGGCTCGGGC GGCTCTGGCGGGTCTCTGCCGGAAAGCGGTGGTGGTTCTGGCGGCTCAGGCGGCTCAGAAAACCTGTACTTC CAGAGCGGTGGCTCCGGCGGTTCTGGTGGCTCCATGGTGTCCAAGGGTGAAGAATTGTTTACCGGTGTGGTG CCGATCCTGGTTGAGTTGGACGGCGATGTTAATGGTCATAAGTTCAGCGTGCGTGGTGAGGGCGAGGGTGAC GCCACCAACGGTAAATTGACCTTGAAGTTCATCTGCACGACTGGTAAGCTGCCAGTGCCGTGGCCGACCTTA GTTACCACGTTAGGTTATGGTGTGGCGTGTTTCGCCCGTTATCCGGACCACATGAAACAGCATGATTTTTTC AAAAGCGCAATGCCGGAAGGTTATGTTCAAGAGCGCACGATTAGCTTTAAGGATGATGGCACGTACAAGACT CGTGCAGAGGTGAAATTCGAGGGAGACACCCTGGTCAACCGCATCGTGCTTAAGGGCATTGACTTCAAAGAG GACGGAAACATTCTTGGCCACAAGTTGGAATACAATTTTAACAGCCATAAGGTGTACATTACCGCGGATAAA CAAAAAAATGGCATCAAAGCGAATTTCAAGACCCGTCATAATGTTGAAGATGGTGGTGTGCAGCTGGCGGAC CACTACCAGCAGAATACCCCGATCGGCGATGGTCCGGTCTTGCTGCCGGATAATCATTATCTGAGTCACCAG AGCAAACTGAGCAAGGACCCGAATGAAAAACGCGATCACATGGTTCTGAAAGAACGTGTTACCGCAGCTGGT ATCACCCATGATATGGATGAATTGTACAAGGGAGGCAGCGGGGGTAGCGGTGGTTCCCTCGCGGAGACCGGT GGGGGAAGCGGTGGCAGCGGCGGCTCTCGTGGCGTACCGCATATTGTGATGGTTGACGCGTATAAACGTTAT AAAGGCGGTTCGGGCGGCTCGGGCGGGAGCCACCACCACCACCACCATGGTGGTTCTGGTGGCTCCGGTGGT AGCGCTGAATACTGCCTGAGCTATGATACCGAGATTTTAACGGTCGAGTACGGCTTTCTGCCGATCGGTAAG ATCGTTGAAGAGCGCATTGAATGCACCGTGTACACGGTTGATAAGAACGGCTTTGTTTACACCCAACCGATT GCACAGTGGCATAACCGCGGTGAGCAAGAAGTTTTTGAGTATTGTCTGGAAGACGGTTCCATAATTCGTGCG ACCAAAGACCACAAGTTCATGACCACCGATGGTCAGATGCTGCCAATCGACGAGATCTTTGAGAGAGGCCTG GACCTGAAACAAGTTGACGGGCTGCCTGCAGCGAACGATGAGAACTATGCTCTGGCTGCG (SEQ ID NO:10) mGreenLantern-S∧(A∨T)3915-P1351WO.UW -47-MGSS-CfaC-GGS(x3)-2A9c-GGS(x3)-TEVc-GGS(x3)-mGreenLantern- GGS(x3)-4S9c-GGS(x3)- SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAGAGCCTTGGTACACAGAACGTCTACGACATCGGT GTAGAGAAAGACCACAACTTCCTGCTGAAGAACGGACTGGTGGCCAGCAATTGTTTTAATGGTGGCTCTGGC GGTTCCGGCGGCAGCCTGGCGGAAACCGGTGGTGGGTCTGGCGGATCCGGCGGATCGGAAAACCTGTATTTC CAGAGCGGAGGCAGTGGTGGTAGCGGCGGTTCGATGGTTAGCAAAGGCGAGGAACTGTTCACCGGTGTCGTG CCGATTCTGGTTGAGCTCGACGGCGACGTTAACGGTCATAAGTTCAGCGTTCGTGGTGAGGGTGAGGGTGAC GCAACCAATGGCAAGTTGACCCTGAAGTTCATCTGCACCACTGGTAAACTGCCGGTTCCGTGGCCGACCTTG GTAACTACGCTGGGCTATGGTGTAGCCTGCTTTGCCCGTTACCCGGATCACATGAAACAACATGATTTCTTT AAATCCGCTATGCCGGAAGGTTACGTGCAAGAGCGCACCATTAGCTTTAAGGACGATGGTACGTATAAAACG CGTGCGGAAGTGAAATTCGAGGGCGACACCCTGGTCAACCGTATTGTTTTAAAGGGCATCGACTTCAAGGAG GACGGTAATATTTTGGGTCATAAACTGGAGTACAATTTCAACAGCCACAAAGTTTATATTACGGCTGATAAA CAAAAAAACGGCATCAAAGCCAACTTTAAAACCCGCCATAACGTCGAGGACGGCGGCGTGCAGTTGGCTGAT CACTACCAGCAAAACACCCCGATTGGCGACGGTCCGGTTTTGCTGCCGGACAATCATTATCTGAGCCATCAG AGCAAGCTCTCCAAGGATCCGAATGAAAAGCGTGATCACATGGTGCTTAAGGAGCGTGTTACCGCAGCGGGT ATCACGCACGATATGGATGAATTGTACAAAGGCGGAAGCGGCGGATCCGGTGGCTCTCTCCCAGAAAGCGGC GGTGGTTCTGGCGGCTCCGGCGGTAGCCGTGGTGTTCCACATATTGTGATGGTGGATGCGTATAAACGTTAC AAGGGTGGTAGTGGCGGTTCTGGTGGCTCGCACCACCATCACCACCACGGTGGTTCCGGCGGCTCTGGTGGT TCAGCGGAGTATTGCCTGAGCTATGACACCGAAATCTTGACCGTTGAATACGGCTTCCTGCCGATTGGTAAA ATCGTGGAAGAGAGAATCGAATGTACCGTGTACACCGTTGATAAAAACGGCTTTGTCTACACTCAACCGATA GCGCAGTGGCATAACCGTGGTGAGCAGGAGGTGTTTGAATACTGCTTAGAGGACGGTAGCATTATCCGCGCG ACCAAGGACCACAAGTTCATGACCACGGATGGTCAGATGCTGCCGATCGATGAGATCTTTGAACGCGGTCTG GATCTGAAGCAAGTTGACGGCCTGCCTGCAGCTAATGATGAGAACTATGCGCTGGCGGCA (SEQ ID NO:11) mGreenLantern-T∧(A∨S) MGSS-CfaC-GGS(x3)-2A9c-GGS(x3)-4S9c-GGS(x3)-mGreenLantern-GGS(x3)- TEVc-GGS(x3)- SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAGAGCCTGGGTACGCAAAACGTGTACGACATCGGC GTGGAGAAGGACCACAACTTTCTGTTGAAGAACGGCCTGGTTGCGAGCAACTGCTTCAATGGCGGCTCCGGT GGATCCGGGGGGTCCCTTGCCGAAACGGGTGGTGGCTCGGGTGGTAGCGGCGGCTCCTTGCCGGAGAGCGGT GGCGGAAGTGGTGGCAGCGGTGGCTCTATGGTGAGCAAAGGTGAAGAGTTGTTTACTGGTGTCGTGCCGATA TTGGTCGAGCTGGACGGTGACGTAAATGGCCATAAATTCAGCGTTCGTGGCGAAGGTGAAGGTGACGCCACC3915-P1351WO.UW -48-AATGGTAAACTGACGTTGAAGTTCATCTGCACCACCGGTAAGTTGCCAGTTCCGTGGCCGACCCTGGTTACC ACTTTGGGTTATGGCGTTGCGTGCTTTGCGCGTTATCCGGATCACATGAAACAGCATGATTTTTTTAAAAGC GCAATGCCGGAAGGCTACGTGCAAGAGCGCACCATCAGTTTTAAGGACGATGGTACATATAAAACCCGTGCA GAAGTGAAATTCGAAGGTGACACCCTCGTGAACCGCATCGTTCTCAAGGGCATCGACTTCAAAGAGGACGGC AACATTCTCGGCCACAAGTTGGAATACAATTTCAACAGCCACAAAGTTTACATCACCGCTGACAAACAAAAA AACGGGATTAAAGCAAACTTCAAGACCCGTCATAATGTCGAGGACGGTGGTGTGCAGCTGGCGGACCACTAC CAACAAAACACCCCGATTGGCGACGGCCCGGTTCTGTTGCCGGATAATCATTACCTGTCCCACCAGAGCAAA CTGAGCAAAGATCCGAATGAAAAACGTGATCACATGGTTCTGAAAGAACGTGTAACTGCGGCCGGTATCACG CATGACATGGATGAACTGTACAAGGGTGGCAGCGGTGGTTCTGGCGGTTCTGAAAACCTGTACTTCCAGAGC GGCGGCAGCGGCGGCTCCGGTGGCAGCCGTGGCGTGCCGCATATTGTTATGGTCGACGCGTATAAGCGCTAC AAGGGCGGTTCTGGTGGTTCGGGTGGCTCACATCACCACCACCACCATGGTGGCAGTGGTGGCTCGGGCGGT TCTGCGGAGTATTGTCTGAGCTATGATACCGAAATCCTGACCGTTGAATATGGTTTCCTGCCAATCGGTAAG ATCGTGGAGGAGCGCATTGAATGTACGGTGTACACCGTTGATAAAAACGGCTTCGTGTATACCCAGCCGATT GCTCAGTGGCATAACCGTGGTGAGCAGGAGGTGTTTGAGTACTGCCTTGAGGACGGTAGCATTATTCGTGCG ACCAAAGACCACAAGTTCATGACCACCGATGGTCAGATGCTGCCGATTGATGAGATCTTTGAAAGAGGTCTG GATCTGAAGCAAGTTGATGGCCTGCCGGCAGCTAATGATGAGAACTATGCGCTGGCTGCG (SEQ ID NO:12) mGreenLantern-A∨(S∧T) MGSS-SnoopTag-GGS(x3)-4S9c-GGS(x3)-mGreenLantern-GGS(x3)-TEVc- GGS(x3)-SnoopCatcher-GGS(x3)-2A9c-GGS(x3)-SpyTag-GGS(x3)LE-6xHis ATGGGAAGTTCAAAACTAGGGGATATAGAATTCATTAAGGTTAACAAAGGCGGTTCTGGCGGTTCC GGTGGCAGCCTGCCGGAGAGCGGCGGTGGCTCGGGTGGAAGCGGCGGCTCCATGGTCAGCAAGGGTGAGGAA TTGTTCACCGGTGTCGTACCAATCCTGGTTGAATTAGATGGCGACGTTAACGGCCACAAGTTCTCCGTGCGC GGTGAGGGTGAGGGTGACGCGACCAACGGCAAATTGACCCTCAAATTCATCTGTACCACCGGTAAACTGCCG GTCCCGTGGCCAACTTTGGTGACGACCCTGGGTTATGGTGTGGCCTGCTTTGCACGTTATCCGGACCACATG AAACAACACGACTTCTTTAAAAGCGCTATGCCGGAAGGCTATGTTCAAGAGCGCACCATTAGCTTTAAAGAT GATGGTACGTACAAGACTCGCGCAGAAGTTAAATTCGAGGGCGATACCTTGGTGAACCGTATTGTGCTGAAA GGCATCGACTTTAAAGAGGACGGTAATATTCTGGGTCACAAACTGGAATATAACTTTAATAGCCATAAGGTG TATATCACTGCGGACAAACAAAAAAACGGCATTAAAGCTAATTTCAAGACCCGTCATAATGTTGAGGACGGT GGCGTGCAACTGGCCGATCACTACCAGCAGAACACCCCGATTGGTGACGGCCCGGTTCTCCTGCCGGATAAT CATTACCTGAGCCACCAGAGCAAATTAAGCAAGGACCCGAATGAAAAACGTGACCACATGGTCCTGAAAGAA CGTGTCACCGCGGCTGGTATCACGCATGATATGGATGAACTGTATAAGGGTGGCTCAGGCGGCTCAGGTGGC TCCGAGAACCTGTACTTCCAAAGCGGCGGCTCCGGCGGCTCTGGGGGTTCCAAACCGCTGCGTGGTGCAGTT3915-P1351WO.UW -49-TTTAGCCTTCAGAAGCAACATCCGGACTACCCGGACATCTATGGTGCGATCGATCAGAACGGTACATACCAG AATGTTCGTACCGGCGAGGATGGTAAGTTGACCTTCAAGAACCTGAGCGATGGTAAGTACCGCCTGTTTGAA AATAGCGAACCGGCTGGTTACAAGCCGGTGCAAAACAAGCCAATCGTTGCGTTTCAGATTGTCAACGGTGAG GTGAGAGACGTGACCAGCATTGTTCCGCAGGATATCCCGGCGACCTACGAATTCACCAATGGCAAGCACTAC ATCACGAACGAGCCGATCCCGCCTAAAGGTGGCTCTGGCGGCTCTGGAGGTTCGTTGGCGGAAACCGGTGGT GGTTCGGGCGGTTCTGGTGGCTCTGCGCATATTGTGATGGTCGATGCGTATAAGCCGACGAAGGGTGGATCC GGCGGCAGCGGTGGTTCCCTCGAGCACCACCACCACCACCAC (SEQ ID NO:13) mGreenLantern- S∨(A∧T) MGSS-SnoopTag-GGS(x3)-2A9c-GGS(x3)-mGreenLantern-GGS(x3)-TEVc- GGS(x3)-SnoopCatcher- GGS(x3)-4S9c-GGS(x3)-SpyTag-GGS(x3)LE-6xHis ATGGGAAGTTCAAAACTAGGGGATATAGAATTCATCAAAGTGAACAAAGGTGGCTCAGGCGGCTCT GGCGGTAGCTTGGCGGAAACCGGTGGTGGTAGTGGCGGCAGCGGAGGCAGCATGGTTAGCAAGGGTGAGGAA CTGTTCACCGGCGTTGTTCCGATCCTGGTCGAGCTGGATGGCGACGTTAACGGTCATAAGTTCAGCGTTAGA GGTGAAGGCGAGGGCGACGCGACGAACGGCAAGTTGACCCTGAAGTTCATCTGCACCACGGGTAAGCTTCCG GTGCCGTGGCCTACCCTGGTGACCACGCTGGGTTACGGGGTGGCGTGTTTTGCACGTTATCCGGATCACATG AAACAACACGACTTCTTCAAGTCAGCTATGCCGGAAGGTTATGTTCAAGAACGTACCATTAGCTTTAAGGAC GACGGTACCTACAAGACCCGCGCGGAAGTGAAATTCGAGGGTGACACCTTGGTTAACCGTATTGTGCTGAAG GGGATCGACTTTAAGGAGGACGGGAATATTTTAGGCCACAAATTGGAATATAACTTCAACAGCCACAAAGTG TATATCACTGCTGACAAACAGAAAAACGGGATCAAAGCTAATTTCAAGACGCGTCATAACGTGGAGGATGGT GGTGTTCAGCTCGCGGATCACTACCAACAGAACACCCCGATCGGTGATGGTCCGGTCCTGCTGCCGGACAAT CATTACCTGTCCCACCAGAGCAAATTAAGCAAAGATCCGAATGAGAAACGTGACCACATGGTACTGAAGGAG CGCGTGACTGCGGCAGGCATTACGCATGATATGGATGAACTCTACAAGGGCGGTAGCGGTGGTAGTGGCGGT TCCGAAAACCTGTATTTCCAAAGCGGTGGTTCCGGCGGTTCGGGCGGCAGCAAGCCGCTGCGTGGTGCAGTT TTTTCTCTGCAGAAACAACATCCGGACTACCCGGACATCTATGGTGCCATTGATCAGAATGGTACATACCAG AACGTACGCACCGGTGAGGACGGCAAACTTACCTTTAAGAATCTGTCTGATGGCAAGTACCGCCTGTTTGAG AATAGCGAACCGGCGGGTTACAAACCGGTCCAAAACAAACCGATCGTGGCCTTTCAAATTGTGAATGGCGAG GTTCGTGATGTTACCAGCATTGTGCCACAGGATATCCCGGCTACCTATGAATTTACCAACGGTAAGCATTAC ATCACGAACGAGCCGATTCCGCCTAAAGGTGGTTCTGGAGGTTCCGGCGGTTCATTGCCAGAAAGCGGCGGT GGCTCTGGCGGTTCGGGCGGCAGCGCGCACATTGTCATGGTTGATGCATATAAGCCGACCAAAGGTGGCAGC GGGGGTTCCGGGGGTTCGCTCGAGCACCACCACCACCACCAC (SEQ ID NO:14) mGreenLantern-T∨(A∧S)3915-P1351WO.UW -50-MGSS-SnoopTag-GGS(x3)-2A9c-GGS(x3)-mGreenLantern-GGS(x3)-4S9c- GGS(x3)-SnoopCatcher- GGS(x3)-TEVc-GGS(x3)-SpyTag-GGS(x3)LE-6xHis ATGGGATCAAGTAAACTAGGGGATATAGAATTCATTAAAGTTAACAAAGGTGGCTCCGGCGGTTCA GGTGGCTCGCTGGCGGAAACCGGCGGCGGTTCCGGCGGTTCTGGCGGGTCTATGGTCAGCAAGGGCGAGGAA CTCTTCACCGGTGTGGTGCCAATCTTGGTGGAACTGGATGGCGACGTTAATGGTCACAAGTTCAGCGTTCGC GGTGAGGGTGAGGGTGACGCGACCAACGGCAAGCTTACCCTGAAGTTCATCTGCACCACCGGTAAATTACCG GTTCCGTGGCCGACGTTGGTTACAACCCTCGGTTACGGCGTAGCATGTTTTGCGCGTTATCCAGACCACATG AAACAGCACGATTTCTTTAAGTCTGCTATGCCGGAGGGTTATGTTCAAGAGCGCACCATTTCGTTCAAGGAT GATGGCACGTACAAGACTCGCGCAGAAGTGAAATTCGAGGGCGACACCCTGGTGAACCGTATCGTGCTGAAG GGTATCGACTTCAAAGAGGACGGAAACATTCTGGGCCACAAACTGGAATATAACTTTAACAGCCACAAGGTG TATATCACCGCGGATAAACAAAAGAACGGCATCAAAGCTAATTTTAAAACGCGTCATAACGTGGAAGACGGC GGCGTGCAACTGGCCGACCATTACCAGCAGAATACCCCGATCGGCGACGGTCCGGTGTTGCTGCCGGATAAT CACTACCTGAGCCATCAGAGCAAGTTGTCCAAGGACCCGAATGAAAAACGTGATCACATGGTGTTAAAAGAA CGTGTTACTGCGGCAGGTATCACCCATGACATGGATGAACTGTACAAGGGTGGTTCCGGTGGTAGCGGCGGT AGCCTGCCGGAGAGCGGTGGCGGTTCTGGCGGTTCAGGTGGTAGCAAGCCGCTGCGTGGTGCCGTTTTTAGC CTGCAGAAACAACATCCGGACTATCCCGACATCTATGGTGCGATTGATCAGAACGGCACCTACCAAAATGTT CGCACCGGTGAAGACGGTAAGTTGACCTTTAAAAATCTGAGCGATGGTAAATACCGTCTGTTTGAGAACTCG GAGCCGGCTGGTTACAAGCCGGTCCAGAACAAGCCGATTGTCGCGTTTCAGATTGTCAACGGTGAGGTTAGA GATGTAACCAGCATTGTTCCGCAGGATATCCCGGCGACGTACGAATTTACCAATGGTAAGCACTACATTACG AACGAACCGATCCCGCCTAAAGGCGGCTCCGGGGGCAGCGGCGGTAGCGAGAACTTGTATTTCCAAAGCGGC GGCTCCGGAGGCAGTGGTGGCAGTGCGCATATTGTTATGGTTGATGCCTATAAGCCGACCAAAGGCGGTTCT GGTGGTTCGGGTGGTTCCCTCGAGCACCACCACCACCACCAC (SEQ ID NO:15) mGreenLantern-A∨S∨T MGSS-mGreenLantern-GGS(x3)-2A9c-GGS(x3)-4S9c-GGS(x3)-TEVc- GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGGTCATCTATGGTAAGTAAAGGAGAAGAGTTGTTTACGGGTGTCGTGCCGATTTTGGTCGAG CTTGATGGTGACGTGAACGGTCATAAGTTCAGCGTTCGTGGTGAAGGTGAAGGCGACGCTACCAATGGTAAA CTGACCTTGAAGTTCATCTGCACCACGGGTAAGTTGCCAGTACCGTGGCCGACCTTGGTCACCACGTTAGGT TATGGTGTGGCGTGTTTTGCCCGTTACCCGGACCACATGAAACAACATGATTTTTTTAAAAGCGCAATGCCG GAAGGTTATGTTCAAGAGCGCACCATTAGCTTTAAGGACGATGGCACCTATAAGACTCGCGCAGAGGTGAAA TTCGAGGGCGACACCCTCGTTAATCGTATTGTTCTGAAGGGTATCGATTTCAAGGAGGACGGTAATATTCTG3915-P1351WO.UW -51-GGTCATAAACTGGAATATAACTTCAACAGCCACAAGGTGTATATCACAGCCGATAAACAAAAAAACGGCATC AAAGCGAATTTTAAGACCCGTCATAACGTTGAGGACGGTGGGGTGCAGCTGGCTGACCACTACCAGCAAAAC ACCCCGATCGGTGACGGCCCCGTACTGCTGCCGGACAACCACTACCTGAGCCACCAGAGCAAACTGAGCAAG GACCCGAATGAAAAACGTGATCACATGGTTCTGAAAGAGCGTGTTACTGCGGCAGGTATTACCCATGATATG GATGAACTGTACAAGGGTGGCAGTGGCGGTTCAGGAGGCTCGCTGGCGGAAACCGGTGGCGGATCCGGCGGT TCTGGCGGGTCGCTGCCGGAGAGCGGTGGCGGCTCCGGGGGCTCTGGCGGTAGCGAAAACCTGTACTTCCAG AGCGGCGGTTCCGGTGGCTCCGGCGGTTCTAGAGGCGTGCCGCATATCGTGATGGTTGATGCGTACAAGCGC TACAAAGGTGGCAGCGGTGGCTCTGGCGGCAGCCACCACCATCACCACCAT (SEQ ID NO:16) mGreenLantern-A∧S∧T MGSS-SnoopTag-GGS(x10)-4S9c-GGS(x10)-DogCatcher-GGS(x10)-SpyTag- GGS(x5)-2A9c-GGS(x5)-mGreenLantern-GGS(x10)-SnoopCatcher-GGS(x10)-TEVc- GGS(x5)-6xHis-GGS(x10)-DogTag ATGGGAAGTTCAAAACTAGGGGATATAGAATTCATCAAAGTTAACAAGGGTGGTTCGGGAGGTAGC GGGGGTAGTGGCGGAAGCGGCGGCTCAGGCGGTTCCGGCGGTAGCGGTGGATCTGGTGGTAGCGGCGGTTCC CTGCCCGAGAGCGGTGGTGGCAGCGGCGGTAGCGGCGGCAGCGGAGGCTCTGGCGGGTCGGGTGGCTCGGGC GGGTCTGGTGGCAGCGGTGGGAGTGGTGGTAGCAAATTAGGCGAGATCGAATTCATCAAGGTGGATAAAACC GATAAAAAACCGCTGCGCGGTGCGGTGTTTAGCCTGCAGAAGCAACACCCGGATTACCCGGACATCTACGGT GCGATTGACCAGAATGGGACGTACCAGGATGTGCGCACTGGTGAGGACGGCAAGTTGACCTTTACCAACCTG TCCGACGGAAAGTATCGTCTTATCGAGAACAGCGAGCCGCCGGGTTATAAGCCGGTTCAAAACAAACCAATC GTTAGCTTTCGTATTGTGGATGGTGAGGTGCGCGACGTGACCTCGATCGTACCGCAGGGTGGGTCGGGCGGT TCCGGGGGTAGCGGCGGATCTGGCGGTTCCGGTGGCTCCGGTGGTAGTGGTGGCAGCGGCGGCTCCGGTGGT TCTGCACATATTGTCATGGTGGACGCATATAAACCGACCAAAGGCGGTAGCGGCGGCTCAGGGGGTTCTGGG GGTAGCGGCGGATCCCTGGCGGAAACCGGCGGTGGTAGCGGTGGTTCCGGAGGTTCCGGCGGCAGCGGCGGC TCGATGGTCAGCAAGGGCGAGGAACTGTTTACCGGTGTCGTGCCAATCCTGGTGGAACTGGATGGGGACGTG AACGGTCATAAGTTCAGCGTTCGTGGTGAAGGTGAGGGTGACGCCACAAATGGCAAATTGACCCTCAAGTTT ATTTGTACCACCGGCAAGTTACCGGTTCCGTGGCCGACGTTGGTTACTACGCTGGGTTACGGCGTGGCGTGC TTCGCGCGTTATCCGGACCACATGAAACAGCATGATTTCTTTAAAAGCGCTATGCCGGAAGGTTACGTTCAA GAGCGTACCATCAGCTTTAAGGATGATGGCACCTACAAGACGCGTGCAGAAGTTAAATTCGAAGGCGATACC TTGGTTAATCGTATCGTTCTGAAGGGCATTGACTTTAAGGAAGACGGCAATATCTTGGGCCACAAACTGGAA TATAACTTCAACAGCCACAAGGTGTACATCACCGCTGACAAACAAAAAAACGGCATCAAAGCGAACTTCAAG ACGCGACATAATGTCGAAGATGGGGGTGTGCAGCTGGCCGATCACTACCAACAGAACACTCCGATTGGCGAC GGTCCGGTTCTCCTCCCGGACAATCACTACCTGTCTCATCAGAGCAAGCTGTCCAAGGATCCGAATGAAAAA3915-P1351WO.UW -52-CGTGACCACATGGTTCTGAAAGAACGTGTTACTGCTGCGGGTATTACGCATGATATGGATGAACTGTACAAA GGTGGCTCCGGTGGTTCGGGCGGTTCTGGTGGCAGCGGTGGTAGCGGGGGGAGCGGAGGCTCCGGTGGTTCC GGCGGCTCGGGCGGTTCCAAGCCGCTGCGTGGTGCGGTTTTTAGCTTGCAAAAGCAACATCCGGATTATCCG GACATCTATGGTGCGATCGACCAGAACGGAACCTACCAAAACGTGCGCACCGGCGAGGACGGCAAGTTGACC TTCAAGAACTTGTCTGATGGAAAGTACCGCCTGTTCGAGAATTCTGAGCCTGCTGGTTACAAACCGGTGCAA AACAAACCGATTGTGGCATTTCAGATTGTTAATGGTGAGGTCAGAGATGTCACCTCCATTGTTCCACAGGAT ATTCCGGCCACCTACGAATTCACCAACGGAAAGCACTATATCACGAACGAACCGATTCCGCCGAAAGGTGGC AGTGGTGGTTCTGGCGGTAGCGGCGGCAGCGGAGGCTCCGGGGGTAGCGGTGGTTCCGGCGGCAGCGGCGGC TCTGGAGGGAGTGAGAACCTGTATTTCCAGTCTGGCGGCTCCGGTGGCTCTGGCGGCAGCGGTGGTTCGGGT GGTTCCCACCACCATCACCACCATGGTGGCAGCGGCGGCTCTGGTGGCAGCGGAGGTAGTGGGGGTTCCGGT GGTTCCGGTGGGTCAGGTGGATCTGGCGGCTCGGGCGGAAGCGACATTCCGGCGACCTACGAGTTCACCGAT GGTAAGCACTATATCACCAATGAGCCGATCCCGCCAAAA (SEQ ID NO:17) Example 5. Mass identification of purified proteins via LC-MS

[0155] Protein identity and purity was confirmed through Liquid Chromatography-Mass Spectrometry (LC-MS). Each sample was filtered (0.22 µm) prior to injection into an LC-MS (AB-Sciex 5600 QTOF) using an inline polymeric reversed- phase column (PL 1912-1503, Agilent). Protein solutions were separated through over the following 15-min linear gradient: 10% acetonitrile for 5 min, then linear gradient from 10% to 90% acetonitrile over 10 min, with mass spectrum scans taken every 1 second in positive mode. The chromatogram was integrated, and the full molecular weight was calculated using Analyst (AB Sciex). Example 6. Plasmid construction and ORF sequences for protease actuator expression

[0156] eSrtA(2A9) (A) and eSrtA(4S9) (S) were gifts from David Liu (Addgene Plasmids #75145 and #75146, respectively), MBP-superTEV (T) from Mark Howarth (Addgene Plasmid #171782), TVMV (V) from David Waugh (Addgene Plasmid #8832), and HRV-3C (C) from Gottfried Otting (Addgene Plasmid #162795). Sequences are given 5’ to 3’. eSrtA(2A9) (A) eSrt(2A9)-GS-6xHis3915-P1351WO.UW -53-ATGCAGGCGAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCGGGTTATATTGAAATTCCGGAT GCGGATATTAAAGAACCGGTGTATCCGGGCCCGGCGACCCGTGAACAGCTGAACCGGGGCGTGTGCTTTCAT GACGAAAACGAAAGCCTGGATGATCAGAACATTAGCATTGCGGGCCATACCTTTATTGATCGTCCGAACTAT CAGTTTACCAACCTGAAAGCGGCGAAACCTGGCAGCATGGTGTATTTTAAAGTGGGCAACGAAACCCGTATA TATAAAATGACCAGCATTCGTAAGGTGCATCCGAACGCGGTGGAAGTGCTGGATGAACAGGAAGGCAAAGAT AAACAGTTGACCCTGGTGACCTGCGATGATTATAACGAAGAAACCGGCGTGTGGGAATCCCGTAAAATTTTT GTGGCGACCGAAGTGAAAGGATCCCACCACCACCACCACCAC (SEQ ID NO:18) eSrtA(4A9) (S) eSrt(4S9)-GS-6xHis ATGCAGGCGAAACCGCAGATTCCGAAAGATAAAAGCAAAGTGGCGGGCTATATTGAAATTCCGGAT GCGGATATTAAAGAACCGGTGTATCCGGGCCCGGCGACCCGTGAACAGCTGGATCGTGGTGTGTGCTTTGTG GAAGAAAACGAAAGCCTGGATGATCAGAACATTAGCATTACCGGCCATACCGCGATTGACCGTCCGAACTAT CAGTTTACCAACCTGAGGGCGGCGAAAAAAGGCAGCATGGTGTATCTTAAAGTGGGCAACGAAACCCGTAAA TATAAAATGACCAGCATTCGTAACGTGAAACCGACCGCGGTGGAAGTGCTGGATGAACAGAAAGGCAAAGAT AAACAGCTGACCCTGGTGACCTGCGATGATTATAACTTTGAAACCGGCGTGTGGGAAACCCGTAAAATTTTT GTGGCGACCGAAGTGAAAGGATCCCACCACCACCACCACCAC (SEQ ID NO:19) MBP-superTEV (T) MBP-6xHis-superTEV-polyR ATGGGAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCT GAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAG AAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGC TACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACC TGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTAT AACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCG AAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGG GGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAA GCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCA GAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACC AGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTG CTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTG ACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAA3915-P1351WO.UW -54-GAGTTGGCGAAAGATCCACGTATTGCCGCCACCATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATC CCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTC GATGAAGCCCTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAACAACCTC GGGATCGAGGGAAGGGGAGATTTTGCAGGTCATCATCATCATCATCATCATGGAGAAAGCTTGTTTAAGGGG CCGCGTGATTACAACCCGATATCGAGCACCATTGTTCATTTGACGAATGAATCTGATGGGCACACAACATCG TTGTATGGTATTGGATTTGGTCCCTTCATCATTACAAACAAGCACTTGTTTAGAAGAAATAATGGAACACTG GTGGTCCAATCACTACATGGTGTATTCAAGGTCAAGAACACCACGACTTTGCAACAACACCTCATTGATGGG AGGGACATGATAATTATTCGCATGCCTAAGGATTTCCCACCATTTCCTCAAAAGCTGAAATTTAGAGAGCCA CAAAGGGAAGAGCGCATAGTCCTTGTGACAACCAACTTCCAAACTAAGAGCATGTCTAGCATGGTGTCAGAC ACTAGTTCGACATTCCCTTCAGGAGATGGCATATTCTGGAAGCATTGGATTCAAACCAAGGATGGGCAGTGT GGCAGTCCATTAGTATCAACTAGAGATGGGTTCATTGTTGGTATACACTCAGCATCGAATTTCACCAACACA AACAATTATTTCACAAGCGTGCCGAAAAACTTCATGGAATTGTTGACAAATCAGGAGGCGCAGCAGTGGGTT AGTGGTTGGCGATTAAATGCTGACTCAGTATTGTGGGGAGGCCATAAAGTTTTCATGGACAAACCTGAAGAG CCTTTTCAGCCAGTTAAGGAAGCGACTCAACTCATGAATCGTCGTCGCCGTCGC (SEQ ID NO:20) HRV-3C (C) 6xHis-NT*-SGGGGS-HRV-3C ATGCATCATCATCATCACCACAGCCATACCACACCGTGGACCAATCCTGGTCTGGCAGAAAACTTT ATGAATAGCTTTATGCAGGGTCTGAGCAGCATGCCTGGTTTTACCGCAAGCCAGCTGGACAAAATGAGCACC ATTGCACAGAGCATGGTTCAGAGCATTCAGAGCCTGGCAGCACAGGGTCGTACCAGTCCGAATGATCTGCAG GCACTGAATATGGCATTTGCAAGCAGCATGGCAGAAATTGCAGCAAGCGAAGAAGGTGGCGGTAGCCTGAGC ACCAAAACCAGCAGCATTGCAAGCGCAATGAGCAATGCATTTCTGCAGACAACCGGTGTTGTTAATCAGCCG TTTATTAACGAAATTACCCAGCTGGTTAGCATGTTTGCACAGGCAGGTATGAATGATGTTAGCGCAGGTAAT AGCGGTGGTGGTGGTAGCGGTCCGAATACCGAATTTGCACTGAGCCTGCTGCGTAAAAACATTATGACCATT ACCACCTCCAAAGGCGAATTTACCGGTCTGGGTATTCATGATCGTGTTTGTGTTATTCCGACACATGCACAA CCGGGTGATGACGTTCTGGTTAATGGTCAGAAAATTCGCGTGAAAGACAAGTATAAACTGGTGGATCCGGAA AACATTAATCTGGAACTGACCGTTCTGACCCTGGATCGTAATGAAAAATTTCGTGATATCCGTGGCTTTATC AGCGAAGATCTGGAAGGTGTTGATGCAACCCTGGTTGTTCATAGCAATAACTTTACCAACACCATCCTGGAA GTTGGTCCGGTTACCATGGCAGGTCTGATTAATCTGAGCAGTACCCCGACCAATCGTATGATTCGTTATGAT TATGCAACCAAAACCGGTCAGTGTGGTGGTGTTCTGTGTGCAACCGGTAAAATCTTTGGCATTCATGTTGGT GGCAATGGTCGTCAGGGTTTTAGCGCACAGCTGAAAAAACAGTATTTCGTGGAAAAGCAG (SEQ ID NO:21) TVMV (V) MBP-GS-TVMVc-6xHis-TVMV3915-P1351WO.UW -55-ATGAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCT GAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAG AAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGC TACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACC TGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTAT AACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCG AAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGG GGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAA GCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCA GAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACC AGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTG CTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTG ACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAA GAGTTGGCGAAAGATCCACGTATTGCCGCCACCATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATC CCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTC GATGAAGCCCTGAAAGACGCGCAGACTAATTCGATCACAAGTTTGTACAAAAAAGCAGGCTCGGAAACCGTG CGTTTCCAGTCTCACCACCATCATCATCACTCTAAAGCTTTGCTGAAGGGCGTGCGCGATTTTAATCCGATC TCTGCTTGCGTATGCCTGCTGGAAAACTCCTCGGATGGTCATAGTGAACGTCTGTTTGGCATTGGTTTTGGC CCGTATATCATTGCCAACCAGCATCTGTTTCGTCGTAACAATGGCGAACTGACCATCAAAACCATGCATGGT GAATTCAAAGTCAAAAACTCTACCCAGCTGCAGATGAAACCGGTTGAAGGCCGTGACATTATCGTTATCAAA ATGGCTAAAGACTTCCCGCCGTTCCCGCAGAAACTGAAATTCCGTCAGCCGACCATCAAAGATCGTGTGTGC ATGGTGTCCACCAACTTTCAGCAGAAAAGCGTCTCGAGCCTGGTGTCTGAATCCTCTCACATTGTGCATAAA GAAGACACTTCTTTCTGGCAGCACTGGATCACCACTAAAGATGGCCAGTGTGGCAGCCCACTAGTTTCCATC ATTGATGGCAACATTCTGGGCATCCACAGCCTGACTCATACCACCAACGGTAGCAACTACTTCGTGGAATTT CCGGAAAAATTCGTGGCGACTTATCTAGATGCCGCGGATGGTTGGTGCAAAAACTGGAAATTCAACGCGGAT AAAATCAGCTGGGGTTCCTTTACCCTGGTTGAAGATGCGCCGGAAGATGACTTCATGGCCAAAAAAACTGTT GCCGCCATCATGGAC (SEQ ID NO:22) Example 7. Mass spectrometric characterization of purified protease actuators

[0157] Liquid chromatography mass spectrometry (LC-MS) analysis was performed for the five protease actuators (A, S, T, V, C). Intended masses and observed LC- MS masses were consistent for each actuator.3915-P1351WO.UW -56-Example 8. Plasmid construction for alternative fluorescent cargo YES gates

[0158] Cloned plasmids [pET-29b(+), with desired sequences inserted between NdeI and XhoI cloning sites] were ordered from GenScript (Piscataway, NJ). The open reading frames (ORFs) of each new construct used for multiplexed hydrogel release studies are given below. Sequences are given 5’ to 3’. All species were designed to contain a SpyTag003 for material tethering and a 6xHis motif for IMAC-assisted purification. mCherry-S MGSS-mCherry-GGS(x3)-4S9c-GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGGAGTTCAATGGTGTCCAAAGGGGAAGAGGACAATATGGCTATCATTAAAGAATTTATGCGT TTCAAAGTACATATGGAAGGAAGCGTTAACGGTCACGAATTCGAGATCGAAGGCGAAGGCGAGGGTCGCCCA TACGAAGGTACGCAGACCGCTAAACTGAAAGTAACCAAAGGCGGACCTTTACCCTTCGCCTGGGACATTCTA AGTCCGCAATTTATGTATGGTAGCAAGGCGTACGTTAAACACCCGGCAGATATCCCAGACTATTTAAAGTTG TCGTTTCCTGAGGGTTTTAAATGGGAGCGAGTGATGAACTTCGAGGATGGGGGCGTGGTCACAGTCACGCAG GATTCGTCGCTGCAGGATGGGGAATTTATTTATAAAGTCAAACTGCGCGGGACTAACTTTCCGTCGGACGGC CCGGTCATGCAAAAAAAAACCATGGGTTGGGAAGCGTCTAGCGAACGTATGTACCCCGAAGATGGGGCATTG AAAGGGGAAATTAAACAACGTCTGAAACTCAAAGATGGCGGACATTATGATGCGGAGGTGAAAACCACTTAT AAGGCGAAAAAACCAGTGCAGCTGCCGGGTGCATATAACGTTAATATTAAACTTGACATCACATCTCATAAT GAAGATTATACCATAGTGGAACAGTACGAACGTGCCGAAGGTCGCCACTCTACGGGCGGCATGGACGAGCTG TACAAGGGCGGTTCCGGCGGTAGTGGTGGAAGCCTGCCGGAAAGCGGCGGAGGTTCAGGAGGTTCCGGCGGC AGTCGGGGCGTACCGCATATTGTTATGGTTGATGCCTATAAGCGCTACAAGGGTGGTTCCGGCGGCAGCGGC GGCTCACACCATCATCACCATCAT (SEQ ID NO:23) mCerulean-T MGSS-mCerulean-GGS(x3)-TEVc-GGS(x3)-SpyTag003-GGS(x3)-6xHis ATGGGATCTTCGATGGTCTCCAAAGGCGAAGAACTGTTCACGGGTGTGGTCCCGATTCTGGTGGAA TTGGATGGTGACGTAAATGGCCATAAGTTTTCGGTGAGCGGCGAAGGCGAAGGCGATGCGACCTATGGCAAG TTAACCCTGAAATTCATCTGTACCACGGGAAAACTACCGGTGCCGTGGCCAACGCTGGTGACCACCCTGACG TGGGGCGTACAGTGCTTTGCTCGTTATCCGGATCACATGAAACAGCACGACTTCTTTAAAAGCGCTATGCCG GAGGGCTACGTGCAGGAACGGACAATCTTTTTTAAAGACGATGGGAATTATAAAACGCGTGCCGAAGTTAAA TTTGAGGGTGATACTCTGGTTAATCGTATCGAACTGAAAGGGATCGATTTCAAAGAGGATGGGAACATACTC GGTCATAAATTGGAGTATAACGCGATTTCGGATAACGTTTATATTACAGCAGACAAACAGAAGAACGGCATT3915-P1351WO.UW -57-AAGGCCAACTTTAAAATCCGCCATAACATTGAAGATGGAAGCGTGCAGCTCGCGGATCACTACCAACAAAAT ACCCCCATCGGCGACGGTCCTGTCCTGCTTCCTGATAATCACTATTTAAGTACTCAAAGTAAACTTTCAAAG GACCCAAACGAAAAACGCGACCACATGGTGTTGCTGGAGTTCGTCACCGCCGCGGGCATTACTTTAGGTATG GATGAACTGTACAAAGGTGGATCAGGTGGTTCCGGGGGCAGCGAAAATCTGTACTTTCAGTCAGGCGGCTCC GGGGGGTCTGGAGGCAGCCGCGGTGTACCGCATATTGTTATGGTTGATGCATACAAGCGATATAAAGGTGGC AGTGGTGGGTCTGGTGGTAGCCATCATCATCACCATCAT (SEQ ID NO:24) Example 9. Mass spectrometry characterization of mCherry-S and mCerulean- T

[0159] LC-MS analysis of extended fluorescent cargo (mCherry-S and mCerulean-T) was performed for hydrogel release studies. Intended masses and observed LC-MS masses were consistent. Example 10. Kinetic analysis of YES-gate cleavage with variable protease and temperature

[0160] mGreenLantern-A, mCherry-S, and mCerulean-T YES-gated protein operators were treated with input protease A, S, and T, respectively. Protease treatments were performed in the following buffer conditions: 20 mM Tris, 50 mM NaCl, 1 mM DTT, 1 mM CaCl2, 18 mM GGG, pH = 7.5. Three different molar ratios of protease to operator were tested at a constant temperature of 4 °C. For A and S, the tested ratios were: 1:500, 1:200, and 1:50. For T, the tested ratios were: 1:50, 1:20, and 1:5. Three different temperatures of 4 °C, 25 °C, and 37 °C were then tested at constant protease to construct ratios of 1:100 forAandSand 1:10 forT.

[0161] Samples were removed from all reactions at 0 minutes, 15 minutes, 1 hour, 3 hours, and 6 hours. The 0-minute samples were removed directly after the addition of all components of the reaction. Samples were quenched by the addition of an equivalent volume of 2x Laemmli buffer containing β-mercaptoethanol and boiling at 95 °C for 7 minutes. Samples were then either used directly for loading on SDS-PAGE gels or frozen at -20 °C until use. Gel densitometry was the performed using Fiji to determine percent cleaved. Briefly, the band intensity for the uncleaved sample per time point was determined, then assuming the first time point (t=0 minutes) equals 0% cleaved, thepercentage of cleavage could then be calculated via Microsoft Excel®.3915-P1351WO.UW -58-Example 11. In-solution treatment of protein library

[0162] Each of the 17 mGreenLantern YES / OR / AND nested protein operators were treated with 23= 8 possible combinations of inputs emanating fromA,S, andT. Protease treatments were performed in the following buffer conditions: 20 mM Tris, 50 mM NaCl, pH = 7.5, 1 mM DTT, 1 mM CaCl2, and 18 mM GGG, supplemented with the input-relevant enzymes. Molar ratios of protease to the appropriate mGreenLantern construct were: 1:100, 1:100, and 1:10 forA,S, andT, respectively. Reaction volumes were adjusted with buffer to compensate for varying volumes of added proteases to equate final protein concentration for gel densitometry. Enzymatic treatments were allowed to proceed for 6 hours at 4 °C, non-kinetically limited endpoints determined to yield full cleavage for all constructs. Reactions were run in triplicate for SDS-PAGE analysis.

[0163] Reactions were quenched by the addition of an equivalent volume of 2x Laemmli buffer containing β-mercaptoethanol and were boiled at 95 °C for 5 min. Samples were then either used directly for loading on SDS-PAGE gels or frozen at -20 °C. Gel densitometry was the performed using Fiji to determine normalized percent of cargo released. Band intensity for the uncleaved and released cargo was determined for each inputcombination, allowing for percent released to be calculated in Microsoft Excel®.Example 12. Expression and purification of SpyCatcher-azide Expression and purification protocol

[0164] E. Coli BL21(DE3) cells were co-transformed with two plasmids: one harboring the SpyCatcher003-6xHis construct with an amber STOP codon in a flexible GS loop, and the other encoding for the appropriate amber suppression machinery (pEvol- pAzFRS.2.t1, a gift from Farren Isaacs, Addgene #73546). The cells were then grown at 37 °C and 220 rpm agitation to an OD of 0.6-0.8 in LB broth supplemented with kanamycin and chloramphenicol. Isopropyl β-D-1-thiogalactopyranoside (IPTG) at a final concentration of 0.5 mM and arabinose at a final concentration of 0.1% were used to induce protein expression, at which point the cultures were moved to a reduced temperature (18 °C) for 18 hours. Cells were then harvested via centrifugation (4,000g, 20 min, 4 °C) and either flash-frozen and stored at -80 °C or used directly.

[0165] Pellets were reconstituted in 40 mL of lysis buffer (20 mM Tris, 50 mM NaCl, pH = 7.5). These were then supplemented with phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1 mM and sonicated on ice (18 min at 30% amplitude3915-P1351WO.UW -59-and 33% duty cycle) (Fisher Scientific, Waltham, MA). After sonication, lysates were centrifuged for 45 min at 11,000g to separate soluble from insoluble fractions. Clarified lysates were loaded onto an ÄKTA Pure 25L FPLC (Cytiva, Marlborough, MA) equipped with a 5mL HisTrap HP column at a flow rate of 5 mL min-1. The HisTrap column was first equilibrated with 5 column volumes of lysis buffer prior to loading. Once all protein was bound, the column was washed with 10+ column volumes of lysis buffer until residual UV read-outs (λ = 280 nm) dropped to 0 absolute units (au). Target protein was then eluted off the column into a 96-well plate by switching to elution buffer (lysis buffer + 250 mM imidazole). The pooled purified protein was dialyzed (20 mM Tris, 50 mM NaCl, pH = 7.5) to remove imidazole (SnakeSkin™ Dialysis Tubing, 3K MWCO, Fischer Scientific, Waltham, MA). After 4+ dialysis bath changes, proteins were spin-concentrated if needed using an Amicon Ultra 15 centrifugation filter (3 kDa MWCO, Sigma-Aldrich, Burlington, MA). The purified solution was supplemented with glycerol (10% of final volume) to act as cryoprotectant, aliquoted into single-use tubes to avoid repeated freeze-thaw cycles, and flash-frozen with liquid nitrogen prior to long-term storage. Plasmid construction for SpyCatcher-azide

[0166] The open reading frames (ORF) of the plasmid required for the generation of SpyCatcher-azide is given below (5’ to 3’). SpyCatcher-azide SpyCatcher003-GSGESGS-AMBER STOP CODON-GSGESGS-6xHis ATGGTGACCACCCTTAGCGGCTTGAGCGGTGAACAAGGCCCGTCCGGTGATATGACCACGGAAGAA GATAGTGCGACCCATATCAAATTCAGCAAACGCGATGAGGACGGCCGGGAGTTAGCTGGTGCGACGATGGAG CTGCGTGATTCATCTGGGAAAACGATTAGTACATGGATCTCGGATGGACACGTGAAAGATTTCTACCTGTAC CCAGGAAAGTATACATTTGTCGAAACCGCAGCTCCGGACGGCTATGAAGTGGCAACTGCCATTACCTTTACC GTTAACGAACAGGGTCAGGTTACTGTAAATGGCGAAGCGACTAAAGGGGACGCCCATACGGGTAGCGGTGAA AGCGGCAGCTAGGGCAGCGGTGAAAGCGGTAGCCACCACCACCACCACCAC (SEQ ID NO:25) Example 13. Development of bead release assay for logical operators

[0167] Magnetic beads functionalized with dibenzocyclooctyne (DBCO) (Vector Laboratories, Newark, CA) were pre-equilibrated in reaction buffer (20 mM Tris, 50 mM NaCl, pH = 7.5, 1 mM DTT, 1 mM CaCl2, 18 mM GGG) and washed. Bead washes consisted of diluting the working bead slurry volume (50-100 µL) in 1 mL of reaction3915-P1351WO.UW -60-buffer. The microcentrifuge tube containing the slurry were placed in a magnetic separation rack to pull the beads towards the tube wall. While tubes were in the slot, fresh reaction buffer was added, and the solution was mixed prior to additional washes using the same method (>5 times).

[0168] After equilibration, beads were pre-reacted with SpyCatcher-azide (200 µL, 1 mg mL-1) for 2 hours at room temperature. Reaction tubes were kept in a stationary tube rotator / mixer throughout the SPAAC conjugation. Afterwards, beads were again washed as described prior to remove any unbound SpyCatcher-azide.

[0169] After SpyCatcher conjugation, the appropriate SpyTagged operator (200 µL, 1 mg mL-1) was added into the solution to conjugate onto the beads through SpyLigation. Reactions were kept in a stationary tube rotator / mixer for 2 hours at room temperature prior to thorough washing to remove any unbound mGreenLantern.

[0170] Once beads were washed, all 8 possible input combinations of A, S, and T were introduced in the same buffer conditions as those used for the in-solution treatments (20 mM Tris, 50 mM NaCl, pH = 7.5, 1 mM DTT, 1 mM CaCl2, 18 mM GGG). Molar ratios of protease to each mGreenLantern (assumed to have conjugated onto the beads at 50% efficiency) construct were: 1:100, 1:100, and 1:10 for A, S, and T, respectively. Treatments were performed for 6 hours at 4 °C. Afterwards, 15 µL was taken from each input condition with a gel-loading pipette and added to a clear-bottom 384 black well plate. Supernatant fluorescence measurements were taken on a BioTek Synergy Microplate Reader (BioTek, Winooski, VT). All conditions were performed in at least three technical triplicates. Fluorescence measurements were normalized by calculating the average fluorescence per input, then the percentage of cargo released was calculated assuming the brightest fluorescence measurement was 100% released. Example 14. Formation of SPAAC-based hydrogels

[0171] Hydrogels (5 µL) were formulated from PEG-tetraBCN (Mn≈ 20,000 Da, 4 mM), triethylene glycol (TEG) diazide (MW = 200.2 g mol-1, 8 mM), SpyCatcher-azide (15 µM), and the set of SpyTagged fluorescent proteins (2 µM each) in gel buffer (20 mM Tris, 50 mM NaCl, 10 mM CaCl2, pH = 7.5). The fluorescent protein constructs and SpyCatcher-azide were first reacted for 30 min at room temperature before the addition of PEG-tetraBCN. After another 30 min, TEG-diazide was also added and the mixture was vortexed. Immediately after the combination of all components, 5 µL volumes of the3915-P1351WO.UW -61-solution were separated into microcentrifuge tubes (1.5 mL) and centrifuged, resulting in cone-shaped gel solutions about 2 mm tall. After waiting 10 min, 75 µL of gel buffer was added to each tube and the formed hydrogels were washed for 72 hours with 3 buffer changes to remove unconjugated fluorescent protein. Example 15. Multiplexed release from polymeric hydrogels

[0172] All treatments were performed at 4 °C in gel buffer (75 µL). Depending on the intended input, samples were treated with a combination of A, S, and T at concentrations of 5 µM, 5 µM, and 7 µM, respectively. DTT (1 mM) was also added to each sample at the same time as the enzymes. After six hours, supernatant fluorescence was measured and used to calculate the corresponding concentrations of released mGreenLantern, mCherry, and mCerulean proteins. Fluorescence normalization was performed as previously stated in the bead release assay method. mGreenLantern was analyzed with λexc.= 498 nm, λem.= 525 nm; mCherry with λexc.= 580 nm, λem.= 610 nm; mCerulean with λexc.= 433 nm, λem.= 475 nm. Example 16. Plasmid construction and sequences for mCerulean-A∧T

[0173] Cloned plasmids [pET-29b(+), with desired sequences inserted betweenNdeI and XhoI cloning sites] were ordered from GenScript®(Piscataway, NJ). The openreading frames (ORFs) of each new construct used for multiplexed hydrogel release studies are given below. Sequences are given 5’ to 3’. All species were designed to contain a SpyTag003 for material tethering and a 6xHis motif for IMAC-assisted purification. mCerulean-A∧T MGSS-CfaC-GGS(x3)-4S9c-GGS(x3)-mCerulean-GGS(x3)-TEVc-GGS(x3)- SpyTag-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAGTCCCTCGGCACCCAAAACGTGTACGATATTGGT GTTGAGAAGGACCACAACTTCTTGTTGAAGAACGGTCTGGTTGCAAGCAATTGTTTCAACGGTGGTTCAGGC GGTTCTGGCGGTTCCCTGCCGGAAAGCGGTGGCGGCTCTGGTGGCTCCGGTGGCTCCATGGTGAGCAAAGGC GAGGAATTGTTTACCGGTGTTGTTCCGATCCTGGTCGAGCTGGATGGCGATGTTAATGGTCACAAGTTCAGC GTGAGCGGTGAAGGTGAGGGTGACGCCACCTATGGCAAGCTGACCCTGAAATTCATCTGCACTACGGGTAAG TTACCAGTCCCGTGGCCGACGCTGGTGACCACCCTGACCTGGGGTGTGCAATGTTTCGCCCGTTATCCGGAT3915-P1351WO.UW -62-CACATGAAACAACATGATTTCTTTAAGTCTGCCATGCCGGAGGGCTACGTTCAAGAACGTACCATCTTTTTC AAGGACGATGGCAACTACAAAACCCGTGCGGAAGTTAAATTTGAAGGTGATACGTTGGTGAACCGTATTGAA TTGAAGGGTATCGACTTCAAAGAAGATGGCAACATTCTGGGTCACAAACTGGAGTACAATGCAATTAGCGAT AACGTTTACATTACCGCGGATAAACAGAAAAACGGCATCAAAGCGAACTTTAAGATCCGCCACAACATTGAG GACGGCAGTGTCCAACTGGCTGACCACTATCAGCAGAATACCCCGATCGGCGACGGTCCGGTGTTGCTTCCA GACAATCATTATCTGAGCACCCAGAGCAAGTTGTCGAAGGACCCGAATGAAAAACGTGATCACATGGTGCTT TTGGAATTTGTTACCGCGGCGGGTATTACCTTAGGTATGGACGAACTGTACAAGGGCGGTTCTGGCGGTTCA GGTGGATCGGAAAATCTGTACTTCCAGAGCGGAGGTAGCGGCGGTAGCGGGGGAAGTGCGCATATTGTGATG GTTGACGCATATAAACCGACAAAAGGTGGTTCCGGCGGCAGCGGTGGTTCCCATCATCACCACCATCACGGC GGTTCCGGGGGCAGCGGTGGATCGGCTGAATACTGCCTGAGCTATGATACCGAAATTCTGACTGTTGAGTAC GGTTTTCTGCCTATCGGCAAGATCGTGGAGGAGCGCATCGAGTGCACCGTCTACACCGTTGATAAAAACGGC TTTGTGTACACTCAGCCGATCGCCCAGTGGCACAACCGCGGTGAACAAGAGGTATTTGAATATTGCCTGGAG GACGGTAGCATCATCCGTGCGACGAAAGACCATAAATTCATGACGACCGATGGTCAGATGCTGCCGATTGAC GAGATCTTCGAGAGAGGCCTCGACCTGAAGCAAGTGGATGGTCTGCCGGCTGCGAATGACGAGAACTATGCA CTGGCGGCT (SEQ ID NO:26) Example 17. Plasmid construction for mGreenLantern-[(A∨S)∧T]∨(C∧V)

[0174] A cloned plasmid [pET-29b(+), with desired sequences inserted between NdeI and XhoI cloning sites] was ordered from GenScript (Piscataway, NJ). The open reading frames (ORFs) of plasmid encoding for [(A∨S)∧T]∨(C∧V)-releasable mGreenLantern is given below. Sequences are given 5’ to 3’. mGreenLantern-[(A∨S)∧T]∨(C∧V) MGSS-CfaC-GGS(x3)-mGreenLantern-GGS(x3)-TEVc-GGS(x3)-SnoopTag- GGS(x3)-HRV-3Cc-GGS(x3)-SpyTag-GGS(x3)-TVMVc-GGS(x3)-6xHis-GGS(x3)- SnoopCatcher-GGS(x3)-4S9c-GGS(x3)-2A9c-GGS(x3)-CfaN-SsrA ATGGGAAGTTCAGTAAAAATTATAAGCAGGAAGAGCCTGGGTACGCAGAATGTGTACGACATCGGT GTGGAGAAGGACCACAATTTCTTATTGAAGAATGGTCTGGTGGCGAGCAACTGCTTTAACGGTGGTTCCGGC GGGTCCGGTGGTAGCATGGTTAGCAAAGGCGAGGAACTGTTCACCGGTGTTGTTCCGATTTTGGTAGAGTTG GACGGCGACGTTAATGGCCATAAGTTCTCTGTTCGTGGTGAGGGCGAAGGTGACGCTACCAACGGTAAGTTG ACGTTGAAGTTTATCTGCACCACCGGTAAATTGCCAGTTCCGTGGCCGACGTTGGTTACCACGCTGGGCTAT GGCGTGGCCTGCTTCGCCCGTTATCCGGACCACATGAAACAACATGATTTTTTCAAGTCTGCTATGCCGGAA GGTTACGTGCAAGAGCGTACCATTAGCTTTAAAGATGATGGCACCTACAAAACCCGCGCTGAAGTCAAATTC3915-P1351WO.UW -63-GAGGGTGATACCCTTGTCAATCGTATCGTTCTGAAGGGCATCGACTTCAAGGAAGATGGTAACATTCTGGGC CACAAGTTGGAATACAATTTCAACAGCCACAAAGTGTATATTACCGCCGATAAACAAAAAAACGGCATCAAA GCAAACTTCAAGACCAGACACAACGTCGAGGACGGCGGCGTTCAACTGGCGGATCACTACCAACAAAACACC CCGATCGGTGATGGTCCGGTTCTGCTGCCGGACAACCATTACCTGAGCCACCAGTCCAAGCTCAGCAAAGAC CCGAATGAGAAGCGTGATCACATGGTACTTAAGGAGCGCGTCACTGCGGCTGGTATCACACATGACATGGAT GAACTGTACAAGGGCGGCAGTGGCGGCAGCGGCGGTTCCGAAAATCTGTACTTCCAAAGCGGCGGATCTGGC GGCAGCGGTGGTAGCAAGCTGGGCGACATCGAGTTCATCAAGGTGAATAAAGGAGGTTCTGGCGGCAGTGGT GGCTCTTTGGAGGTTCTATTCCAAGGTCCGGGTGGCTCTGGTGGTAGCGGTGGCTCGGCCCATATTGTTATG GTTGATGCGTATAAGCCCACCAAAGGTGGCTCGGGTGGTAGCGGTGGTTCGGAAACGGTGCGCTTTCAGAGC GGCGGTTCTGGCGGCAGCGGTGGTAGCCACCACCACCACCATCACGGCGGTAGCGGCGGTTCGGGCGGTTCA AAACCGCTGCGTGGTGCAGTTTTTAGCCTGCAGAAACAACATCCGGATTATCCGGACATCTACGGTGCGATT GACCAGAACGGCACGTACCAGAACGTCCGCACCGGTGAGGACGGTAAACTGACCTTTAAGAACCTGAGTGAT GGTAAATATCGTCTCTTTGAGAACTCAGAGCCGGCGGGTTACAAGCCGGTGCAGAACAAGCCGATCGTGGCG TTTCAAATTGTGAACGGCGAGGTTCGTGATGTGACTTCAATTGTCCCGCAGGATATTCCGGCGACCTATGAA TTTACTAATGGTAAGCATTACATCACCAACGAACCGATCCCGCCTAAAGGTGGGAGCGGTGGTAGCGGCGGT TCCCTGCCTGAAAGCGGCGGTGGCTCGGGGGGCTCCGGTGGGTCCTTGGCGGAAACCGGTGGTGGCTCCGGG GGAAGCGGGGGCTCCGCTGAATACTGTCTGAGCTATGACACCGAAATTCTGACGGTGGAGTACGGCTTTCTG CCAATAGGCAAGATCGTGGAAGAGCGCATTGAATGTACTGTCTATACCGTGGATAAAAACGGCTTCGTGTAC ACCCAGCCGATTGCACAGTGGCATAACCGTGGTGAGCAGGAGGTGTTCGAATACTGCCTGGAGGACGGTTCT ATCATTCGTGCGACGAAAGACCATAAATTCATGACCACAGATGGCCAGATGCTGCCAATCGACGAGATCTTT GAACGCGGTCTCGACCTGAAACAGGTTGACGGTCTGCCGGCGGCAAATGATGAAAACTATGCACTGGCGGCG (SEQ ID NO:27) Example 18. Development of 5-input bead release assay for mGreenLantern- [(A∨S)∧T]∨(C∧V)

[0175] Beads were pre-equilibrated in reaction buffer, washed extensively, and functionalized with SpyCatcher-azide and the mGreenLantern-[(A∨S)∧T]∨(C∧V) cargo.

[0176] Once beads are washed after mGreenLantern operator tethering, all 32 possible input combinations of A, S, T, V, and C were introduced in the same buffer conditions as those used for the in-solution treatments (20 mM Tris, 50 mM NaCl, pH = 7.5, 1 mM DTT, 1 mM CaCl2, 18 mM GGG). OperatorsAandSwere introduced in a 1:25 protease:operator molar ratio, T and V at 1:10, and C at 1:20. After a 2-hour treatment at room temperature, 15 µL was taken from each input condition with a gel-loading pipette3915-P1351WO.UW -64-and added to a 384-well clear bottomed black plate. Supernatant fluorescence measurements were taken on a BioTek Synergy Microplate Reader (BioTek, Winooski, VT). Fluorescence normalization was performed as previously stated in the bead release assay method. All conditions were performed in at least three technical triplicates. Example 19. Protease mammalian cell biocompatibility assay

[0177] Human embryonic kidney (HEK293T) cells were seeded in triplicate in a white walled, clear bottom 96 well plate in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S).The following day, the media was supplemented with 1mM DTT, 1mM CaCl2, 18mMGGG, 1µM of either A, S, or T, a combination of all conditions, or nothing added. Additionally, a dead control prepared by incubating cells with 50% DMSO. After 29 hours, Live / Dead staining reagents [2µM Calcein AM and 4µM of ethidium homodimer-1 (EtHD- 1)] were incubated with the cells for an hour. Cells were then imaged on a Leica Stellaris5®confocal microscope at 10x magnification. Fiji®was first used to split channels percondition, then images with dense cell populations (separate channels: one channel for all conditions, and another channel for the dead control) were fed through a Cell Profiler cell counting pipeline. For images with little to no cells (channel for all conditions, excludingthe dead condition), Fiji®was used to count dead cells (channel for all conditions, exceptthe other channel for the dead condition). Percent live was then calculated in MicrosoftExcel®.Example 20. Plasmid construction for Boolean HER2 nanobody membrane labeling

[0178] The HER2 Nanobody cloned plasmid [pET-29b(+), with desired sequences inserted between NdeI and XhoI cloning sites] was ordered from GenScript (Piscataway, NJ). The fluorescent tag (eGFP-SpyCatcher) ordered as a gBlock encoding for eGFP-SpC-6xHis, then inserted into a pET21b backbone using Gibson assembly (gBlock and oligos from IDT). The open reading frames (ORFs) of the HER2 Nanobody and eGFP catcher are given below. Sequences are given 5’ to 3’. HER2NB-C∧(A∨T)-SpyTag MGSS-CfaC-GGS(x3)-2A9c-GGS(x6)-TEVc-GGS(x3)-HER2NB-GGS(x3)- HRV-3Cc-GGS(x3)- SpyTag003-GGS(x3)-6xHis-GGS(x3)-CfaN-SsrA3915-P1351WO.UW -65-ATGGGAAGTTCAGTAAAAATTATATCTAGGAAGAGCCTGGGTACTCAAAACGTGTACGACATCGGC GTTGAAAAAGACCATAATTTTTTGCTGAAGAACGGTCTGGTGGCTAGCAATTGTTTCAACGGTGGCTCGGGC GGTTCTGGCGGCAGCTTGGCGGAAACCGGTGGCGGCTCCGGCGGCAGTGGAGGCTCCGGCGGCTCCGGCGGG TCGGGTGGTAGCGAAAACCTGTATTTCCAAAGCGGCGGTTCGGGCGGTAGTGGCGGTTCCGTCCAACTGCAG GAGAGCGGCGGTGGCTTGGTTCAGCCGGGCGGTTCTCTGAGATTAAGCTGTGCCGCGAGCGGTTTCACGTTT GGTGATAGCGGCATGGGTTGGTATCGTCAGGCACCGGGAAACGAGTGCGAATTGGTCAGCTCAGTTAGCTCG GATGGTTCCACCTATTACGCGGACAGCGTTAAAGGCCGTTTTACCATTAGCCAGGATAATGCGAAAAACACC GTCTACTTGCGTATGAATAGCCTTAAGCCGGAAGATACGGCTGTTTACTACTGCGCGGCTGATGATCACAAG TACGAACTGGGTACGTGCGAGGCACTGGACTATTGGGGTCGCGGTACACAAGTGACCGTTAGCAGCGGCGGT AGCGGCGGGTCGGGTGGCTCATTGGAGGTGCTGTTTCAAGGTCCGGGTGGATCCGGTGGATCTGGCGGTAGC GCGCATATCGTGATGGTGGACGCATATAAACCGACCAAGGGTGGCTCCGGCGGTTCCGGCGGCAGCCATCAC CACCACCACCATGGTGGTTCCGGTGGTAGCGGTGGTTCTGCCGAATACTGCCTGAGCTATGACACTGAAATC CTGACCGTGGAGTATGGTTTCCTGCCAATCGGTAAAATCGTAGAGGAACGCATTGAATGTACCGTTTACACC GTGGACAAAAACGGCTTCGTGTACACGCAACCGATTGCGCAGTGGCATAACCGCGGTGAACAGGAGGTTTTT GAGTACTGCCTGGAGGACGGCTCCATTATTCGTGCTACCAAGGACCACAAATTCATGACCACCGATGGTCAG ATGCTGCCGATCGATGAGATCTTCGAGCGTGGTCTTGACCTGAAGCAGGTTGACGGTCTGCCGGCAGCTAAT GATGAGAACTATGCGCTGGCGGCGTAA (SEQ ID NO:28) eGFP-SpyCatcher MRGS-6xHis-GS(x6)-eGFP-GS(x5)-SpyCatcher003 ATGAGAGGATCGCATCACCATCACCATCACGGATCTGGCTCCGGAAGCGGAAGTGGTTCTGGTAGC GTCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGAC GTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAG TTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAG TGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC ACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTG GAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAG AAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTAC AAGGGATCTGGCTCCGGAAGCGGAAGTGGTTCTGTGACCACCCTTAGCGGCTTGAGCGGTGAACAAGGCCCG3915-P1351WO.UW -66-TCCGGTGATATGACCACGGAAGAAGATAGTGCGACCCATATCAAATTCAGCAAACGCGATGAGGACGGCCGG GAGTTAGCTGGTGCGACGATGGAGCTGCGTGATTCATCTGGGAAAACGATTAGTACATGGATCTCGGATGGA CACGTGAAAGATTTCTACCTGTACCCAGGAAAGTATACATTTGTCGAAACCGCAGCTCCGGACGGCTATGAA GTGGCAACTGCCATTACCTTTACCGTTAACGAACAGGGTCAGGTTACTGTAAATGGCGAAGCGACTAAAGGG GACGCCCATACGGGTTCTTAA (SEQ ID NO:29) Example 21. Boolean HER2 nanobody membrane labeling Expression and Purification

[0179] The plasmid for the logically releasable HER2 nanobody (HER2Nb- C∧(A∨T)-SpyTag) was transformed into SHuffle T7 Competent E. coli, while the plasmid for eGFP-SpyCatcher was transformed into BL21(DE3) E. Coli (NEB). Both proteins were expressed following an established protocol, except the HER2Nb was incubated at 30 °C in LB media until an OD of 0.4-0.5. IPTG (0.4 mM) was added to induce protein expression and incubated overnight at 16°C. Purification with an endotoxin wash (standard lysis buffer supplemented with 0.1% triton X-114) and dialysis protocols were followed. SpyLigation and Sample Preparation

[0180] Protein concentration of HER2Nb-C∧(A∨T)-SpyTag and eGFP- SpyCatcher were determined by NanoDrop (A280 absorbance), then mixed at a 1-to-1 molar ratio. The solution pH was adjusted between 7.0 to 8.0, then sterile filtered. The reaction occurred for 14-16 hours at 4 °C on a rocker. The HER2Nb now tagged with eGFP[denoted HER2Nb-C∧(A∨T)-eGFP] was supplemented with 1mM of DTT, 1mM of CaCl2and 18mM of GGG, then treated with 23 = 8 possible combinations of inputs emanating from A, C, and T. Molar ratios of protease added were: 1:100, 1:50, and 1:50 for A, C, and T, respectively. Enzymatic treatments were allowed to proceed for 6 hours at 4 °C, non- kinetically limited endpoints determined to yield full cleavage for all constructs. HER2Nb-C∧(A∨T)-eGFP Membrane Labeling- Confocal Imaging

[0181] SK-BR-3 breast cancer cells overexpressing HER2 receptors were seeded overnight on an eight-chamber glass slide (ibidi) in DMEM supplemented with 1% P / S and 10% FBS. The wells were washed once with cold PBS, then 100mM of protease treated HER2Nb-C∧(A∨T)-eGFP and 1:1500 dilution of Hoechst 33342 in PBS was incubated with the cells for 1 hour at 4°C. The cells were washed twice with cold PBS, then ready toimage on a Leica Stellaris 5®confocal microscope at 10x magnification.HER2Nb-C∧(A∨T)-eGFP Membrane Labeling- Flow Cytometry3915-P1351WO.UW -67-

[0182] SK-BR-3 were passaged and resuspended at 1x106cells / mL inFluorescence-activated cell sorting (FACS) buffer (PBS, 1% BSA). The cells were washed once with cold FACS buffer, then 100mM of protease treated HER2Nb-C∧(A∨T)-eGFP in FACS buffer was incubated with the cells for 1 hour at 4°C. The cells were washed twice with cold FACS buffer, then eGFP positive events were collected on a BDFACSSymphony A3®analyzer (BD Bioscience®; San Jose, CA).Example 22. Intracellular Boolean logic membrane labeling and protease treatments

[0183] HEK293T cells were plated at ~40% confluency and allowed to adhere to tissue culture plastic overnight. Fresh media was added, and then HEKs were transfected with envelope plasmid pMD2.G (Addgene #12259), packaging plasmids pMDLG / pRRE (Addgene #12251) and pRSV-REV (Addgene #12253), and the mGreenLantern-C∧(T∨V)- CAAX using Lipofectamine 2000 (Invitrogen). Cells were cultured for 2 days post- transfection, and virus-laden media was harvested. Viral media was filtered (0.45 μm) and tested using lentiviral titration card (ABM Biologics). Active lentivirus was concentrated by mixing viral media with 4X lentiviral concentration solution (40% w / v PEG-8000, 1.2 M NaCl), vigorously shaking for 60 seconds, and agitated overnight at 4 °C. On the following day, flocculated lentiviral particles were pelleted at 1600×g for 60 minutes at 4 °C, and supernatant was aspirated. Pellet was resuspended at 10X relative to initial viral media volume in PBS. This concentrated viral stock was stored at -80 °C until use.

[0184] Fresh HEK293T cells were plated at ~40% confluence in a 35 mm dish (Fisher Scientific, Waltham, MA). After allowing the cells to adhere, media was supplemented with concentrated lentivirus obtained in the previous step and incubated overnight. The following day, mGreenLantern production was confirmed via confocal microscopy (Leica, Wetzlar, Germany) and viral-laden media was removed and bleached to destroy any pendant lentiviral particles. After replenishing the well with fresh DMEM, cells were selected in 10 µg mL-1 of Puromycin (Fisher Scientific, Waltham, MA). Cells were incubated in selection media for 24 hours, after which non-adherent cells were removed and fresh DMEM was used to replenish the well and allow the surviving transgenic cells to recover and expand. mGreenLantern expression was optimized later via fluorescence assisted cell sorting on a Symphony A6 (BD BioSciences, Franklin Lakes, NJ).3915-P1351WO.UW -68-

[0185] Protease treatment was performed via transient transfection of plasmids containing protease(s) on a polycistronic expression cassette with a C-terminally expressed mCherry cassette to assay transfection efficiency. mGreenLantern expressing HEK293Ts were transfected with Lipofectamine 3000 (Invitrogen, Waltham, MA) and allowed to recover for 24 hours. Cells were then imaged using confocal microscopy (Leica, Wetlzar, Germany) and saved images were cropped such that one cell expressing both mGreenLantern and mCherry were present per image. Single-cell images were processed using CellProfiler.2Regions of interest were manually determined and membrane / cytosolic localization of mGreenLantern was reported as a radial distribution function relative to the center of a cell-containing region of interest. The outermost 20.8% of the object was considered to be the membrane and the interior remainder was considered to be the cytosol. Example 23. Plasmid construction for intracellular Boolean logic membrane labeling and protease treatments

[0186] The Boolean logic construct for membrane labeling was inserted into a pGenLenti backbone and inserted between the BamHI and PmeI cloning sites. All cloned plasmids for protease treatment were designed in a pcDNA3.1(+) backbone with desired sequences inserted between HindIII and EcoRI cloning sites. All plasmids were ordered from GenScript (Piscataway, NJ). The open reading frames (ORFs) of each new construct used for intracellular Boolean logic membrane labeling and protease transfection studies are given below. Sequences are given 5’ to 3’.

[0187] The Boolean logic construct for membrane labeling contains a CAAX tag to transport the protein to the cellular membrane. All protease plasmids for transfection contain P2A motifs to cleave apart proteases and an mCherry reporter protein. mGreenLantern-C∨(T∧V)-CAAX MGSS-SnoopTag-GGS(x3)-TVMVc-GGS(x3)-mGreenLantern-GGS(x3)-TEVc- GGS(x3)-SnoopCatcher-GGS(x3)-HRV-3Cc-GGS(x3)-CAAX* ATGGGCAGTTCTAAGCTGGGAGATATCGAGTTCATCAAGGTGAACAAGGGCGGCTCCGGAGGTTCA GGCGGCTCTGAAACCGTGCGCTTCCAGAGCGGCGGCTCAGGAGGCAGCGGAGGCTCTGTGTCCAAGGGCGAG GAACTGTTCACCGGCGTGGTGCCCATCCTGGTTGAGCTGGACGGCGACGTGAACGGCCACAAGTTTAGCGTG CGGGGCGAAGGCGAGGGCGACGCCACAAACGGCAAGCTGACCCTGAAATTCATCTGTACAACCGGCAAACTG CCTGTGCCATGGCCTACACTGGTCACAACACTGGGCTACGGCGTGGCCTGCTTCGCCAGATACCCCGATCAC3915-P1351WO.UW -69-ATGAAACAGCATGACTTTTTCAAAAGCGCCATGCCGGAAGGCTACGTGCAGGAGCGGACCATCAGCTTCAAG GACGACGGAACCTACAAGACCAGAGCCGAGGTTAAGTTCGAGGGCGACACCCTGGTGAATAGAATCGTGCTG AAGGGCATCGACTTCAAGGAAGATGGAAATATCCTGGGCCATAAGCTGGAATACAACTTCAACAGCCACAAG GTGTACATCACCGCCGATAAGCAGAAAAACGGCATTAAGGCCAATTTCAAGACCCGGCACAACGTGGAAGAT GGCGGGGTGCAGCTGGCCGACCACTACCAGCAGAATACCCCTATCGGCGACGGCCCTGTCCTGCTGCCCGAC AACCACTACCTGAGCCACCAGAGCAAGCTGAGCAAAGATCCTAACGAGAAGCGGGACCACATGGTGCTGAAG GAAAGAGTGACCGCCGCCGGCATCACCCACGACATGGACGAGCTGTACAAGGGCGGCAGCGGCGGAAGCGGA GGCAGCGAGAACCTGTACTTCCAATCTGGCGGCAGCGGCGGAAGCGGCGGCTCCAAGCCTCTGAGAGGCGCT GTGTTCAGCCTGCAGAAGCAACACCCCGACTACCCTGATATCTACGGCGCCATCGACCAGAACGGCACATAT CAGAATGTGAGAACCGGAGAAGATGGCAAGCTCACCTTCAAGAACCTGAGCGACGGCAAATACAGACTGTTT GAGAACAGCGAGCCCGCTGGCTATAAGCCTGTGCAAAACAAGCCTATCGTGGCCTTCCAGATCGTGAACGGC GAGGTCAGAGATGTGACCAGCATCGTGCCTCAGGACATCCCTGCTACATACGAGTTTACCAACGGAAAGCAC TACATCACAAACGAGCCTATTCCTCCAAAGGGCGGCAGCGGCGGCAGCGGCGGCAGCCTGGAAGTGCTGTTC CAGGGCCCCGGCGGCTCTGGAGGATCTGGCGGCAGCATGAGCAAGGACGGCAAGAAAAAGAAGAAGAAATCT AAGACAAAGTGCGTGATCATGTGA (SEQ ID NO:30) C Protease & mCherry MGSS-HRV-3C Protease-GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTGGACCTAACACCGAGTTCGCCCTGAGCCTACTGAGAAAGAACATCATGACCATC ACCACCAGCAAGGGCGAGTTCACCGGCCTGGGCATCCACGACAGAGTGTGCGTGATCCCCACCCATGCCCAG CCGGGAGATGATGTGTTGGTGAACGGCCAAAAAATCCGGGTGAAGGACAAGTACAAGCTGGTGGACCCTGAA AACATCAACCTGGAACTGACCGTGCTGACACTGGATCGGAATGAAAAATTTCGAGACATCAGAGGCTTTATC TCTGAGGACCTGGAAGGCGTCGACGCCACACTGGTGGTCCACAGCAACAACTTTACAAATACCATCTTGGAG GTGGGCCCTGTGACCATGGCTGGCCTGATCAACCTGTCTAGCACACCTACAAACAGAATGATCCGATACGAC TACGCCACAAAAACCGGCCAGTGTGGCGGAGTGCTGTGCGCCACTGGCAAAATTTTCGGCATTCACGTGGGA GGCAATGGCAGACAGGGCTTCTCTGCCCAGCTGAAGAAGCAGTACTTCGTGGAAAAGCAGGGCTCCGGCAGT GCCACAAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCGGGAGCGGCTCCGTG AGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAAGGCAGC GTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGACCCTACGAGGGAACACAGACCGCCAAG CTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATCCTGAGCCCTCAGTTCATGTATGGCAGC AAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAACTGTCTTTTCCTGAGGGATTCAAGTGG GAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACCCAGGACAGCTCCCTCCAGGACGGCGAG TTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGACGGCCCCGTGATGCAGAAGAAAACAATG3915-P1351WO.UW -70-GGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCCCTGAAAGGCGAAATCAAACAAAGACTG AAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACCTACAAGGCCAAAAAGCCTGTGCAGCTG CCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAG TACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAGCTGTACAAGTGA (SEQ ID NO:31) T Protease & mCherry MGSS-SuperTEV-GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTGGGGAGAGCCTGTTTAAAGGCCCCAGAGATTATAATCCCATCAGCTCCACCATC GTGCACCTGACAAACGAGAGCGACGGACACACCACAAGCCTGTACGGAATCGGCTTTGGACCTTTCATCATT ACCAACAAGCACCTGTTCAGACGGAACAACGGCACGCTGGTGGTGCAGAGCCTGCACGGCGTTTTTAAGGTG AAGAACACCACCACCCTGCAGCAGCATCTGATCGACGGAAGAGACATGATCATCATCAGGATGCCTAAGGAC TTCCCACCTTTCCCTCAGAAGCTGAAATTCAGGGAGCCTCAGAGAGAGGAGCGGATCGTCCTGGTGACCACC AACTTCCAGACAAAAAGCATGAGCTCTATGGTGTCCGATACAAGCAGCACCTTCCCTTCCGGCGACGGCATC TTCTGGAAGCACTGGATCCAAACCAAAGATGGCCAGTGCGGCTCTCCCCTGGTGAGCACTAGAGATGGCTTC ATCGTGGGCATCCACAGCGCTAGCAACTTCACCAACACTAACAACTACTTTACCAGCGTTCCTAAGAATTTC ATGGAACTGCTGACCAACCAGGAAGCCCAACAATGGGTGAGCGGTTGGCGGCTGAACGCCGATTCTGTGCTG TGGGGAGGACACAAGGTATTCATGGATAAGCCTGAAGAACCTTTCCAGCCTGTCAAAGAGGCCACACAGCTG ATGAACCGGAGAAGAAGAAGAGGCAGCGGCAGCGCCACCAATTTCAGCCTGCTGAAGCAGGCTGGCGACGTG GAGGAAAACCCAGGCCCAGGTAGCGGCTCGGTGAGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAG TTCATGAGATTCAAGGTGCACATGGAAGGCAGCGTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAA GGCAGACCCTACGAGGGAACACAGACCGCCAAGCTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGG GACATCCTGAGCCCTCAGTTCATGTATGGCAGCAAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTAC CTGAAACTGTCTTTTCCTGAGGGATTCAAGTGGGAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACC GTGACCCAGGACAGCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCC TCTGACGGCCCCGTGATGCAGAAGAAAACAATGGGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGAC GGAGCCCTGAAAGGCGAAATCAAACAAAGACTGAAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAG ACAACCTACAAGGCCAAAAAGCCTGTGCAGCTGCCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACC TCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATG GACGAGCTGTACAAGTGA (SEQ ID NO:32) V Protease & mCherry MGSS-TVMV-GS(x2)-P2A-GS(x2)-mCherry*3915-P1351WO.UW -71-ATGGGCTCCTCTAGCAAGGCTCTGCTGAAAGGAGTGCGGGATTTCAACCCCATCAGCGCATGCGTG TGCCTGCTGGAGAACAGCTCTGACGGACACTCTGAAAGACTCTTCGGCATCGGCTTCGGCCCTTACATCATC GCCAATCAGCACCTTTTCCGGAGAAACAACGGCGAGCTGACAATCAAGACCATGCACGGAGAGTTCAAGGTG AAGAATAGTACACAGCTGCAGATGAAACCCGTTGAGGGACGGGACATCATCGTGATCAAGATGGCTAAGGAC TTCCCTCCATTCCCCCAGAAACTGAAGTTCCGGCAGCCTACCATCAAGGATAGAGTGTGTATGGTGAGCACC AACTTCCAGCAGAAGAGCGTATCCAGCCTGGTGTCCGAAAGCAGTCACATCGTGCATAAGGAAGACACCTCC TTCTGGCAGCACTGGATCACCACCAAGGACGGACAATGTGGCTCTCCTCTGGTGAGCATCATTGACGGCAAC ATCCTGGGCATCCACTCTCTCACACATACCACAAATGGGAGCAACTACTTCGTGGAGTTCCCTGAAAAGTTT GTGGCCACGTACCTGGATGCCGCTGACGGCTGGTGCAAGAATTGGAAGTTCAACGCCGATAAGATCAGCTGG GGCAGCTTCACCCTGGTGGAGGATGCCCCTGAGGATGACTTCATGGCCAAGAAGACCGTCGCCGCCATCATG GACGGTAGCGGCAGCGCCACAAACTTCAGCCTCCTCAAGCAGGCTGGGGACGTTGAGGAGAACCCTGGCCCC GGCTCCGGCTCTGTGAGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATGAGATTCAAGGTG CACATGGAAGGCAGCGTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGACCCTACGAGGGA ACACAGACCGCCAAGCTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATCCTGAGCCCTCAG TTCATGTATGGCAGCAAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAACTGTCTTTTCCT GAGGGATTCAAGTGGGAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACCCAGGACAGCTCC CTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGACGGCCCCGTGATG CAGAAGAAAACAATGGGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCCCTGAAAGGCGAA ATCAAACAAAGACTGAAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACCTACAAGGCCAAA AAGCCTGTGCAGCTGCCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACCTCCCACAACGAGGACTAC ACCATCGTGGAGCAGTACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAGCTGTACAAGTGA (SEQ ID NO:33) T and C Proteases & mCherry MGSS-SuperTEV-GS(x2)-P2A-GS(x2)-HRV-3C-GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTGGGGAGAGCCTGTTTAAAGGCCCCAGAGATTATAATCCCATCAGCTCCACCATC GTGCACCTGACAAACGAGAGCGACGGACACACCACAAGCCTGTACGGAATCGGCTTTGGACCTTTCATCATT ACCAACAAGCACCTGTTCAGACGGAACAACGGCACGCTGGTGGTGCAGAGCCTGCACGGCGTTTTTAAGGTG AAGAACACCACCACCCTGCAGCAGCATCTGATCGACGGAAGAGACATGATCATCATCAGGATGCCTAAGGAC TTCCCACCTTTCCCTCAGAAGCTGAAATTCAGGGAGCCTCAGAGAGAGGAGCGGATCGTCCTGGTGACCACC AACTTCCAGACAAAAAGCATGAGCTCTATGGTGTCCGATACAAGCAGCACCTTCCCTTCCGGCGACGGCATC TTCTGGAAGCACTGGATCCAAACCAAAGATGGCCAGTGCGGCTCTCCCCTGGTGAGCACTAGAGATGGCTTC ATCGTGGGCATCCACAGCGCTAGCAACTTCACCAACACTAACAACTACTTTACCAGCGTTCCTAAGAATTTC3915-P1351WO.UW -72-ATGGAACTGCTGACCAACCAGGAAGCCCAACAATGGGTGAGCGGTTGGCGGCTGAACGCCGATTCTGTGCTG TGGGGAGGACACAAGGTATTCATGGATAAGCCTGAAGAACCTTTCCAGCCTGTCAAAGAGGCCACACAGCTG ATGAACCGGAGAAGAAGAAGAGGCAGCGGCAGCGCCACCAATTTCAGCCTGCTGAAGCAGGCTGGCGACGTG GAGGAAAACCCAGGCCCAGGTAGCGGCTCGGGACCTAACACCGAGTTCGCCCTGAGCCTACTGAGAAAGAAC ATCATGACCATCACCACCAGCAAGGGCGAGTTCACCGGCCTGGGCATCCACGACAGAGTGTGCGTGATCCCC ACCCATGCCCAGCCGGGAGATGATGTGTTGGTGAACGGCCAAAAAATCCGGGTGAAGGACAAGTACAAGCTG GTGGACCCTGAAAACATCAACCTGGAACTGACCGTGCTGACACTGGATCGGAATGAAAAATTTCGAGACATC AGAGGCTTTATCTCTGAGGACCTGGAAGGCGTCGACGCCACACTGGTGGTCCACAGCAACAACTTTACAAAT ACCATCTTGGAGGTGGGCCCTGTGACCATGGCTGGCCTGATCAACCTGTCTAGCACACCTACAAACAGAATG ATCCGATACGACTACGCCACAAAAACCGGCCAGTGTGGCGGAGTGCTGTGCGCCACTGGCAAAATTTTCGGC ATTCACGTGGGAGGCAATGGCAGACAGGGCTTCTCTGCCCAGCTGAAGAAGCAGTACTTCGTGGAAAAGCAG GGCTCCGGCAGTGCCACAAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCGGG AGCGGCTCCGTGAGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATGAGATTCAAGGTGCAC ATGGAAGGCAGCGTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGACCCTACGAGGGAACA CAGACCGCCAAGCTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATCCTGAGCCCTCAGTTC ATGTATGGCAGCAAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAACTGTCTTTTCCTGAG GGATTCAAGTGGGAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACCCAGGACAGCTCCCTC CAGGACGGCGAGTTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGACGGCCCCGTGATGCAG AAGAAAACAATGGGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCCCTGAAAGGCGAAATC AAACAAAGACTGAAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACCTACAAGGCCAAAAAG CCTGTGCAGCTGCCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACCTCCCACAACGAGGACTACACC ATCGTGGAGCAGTACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAGCTGTACAAGTGA (SEQ ID NO:34) V and C Proteases & mCherry MGSS- TVMV-GS(x2)-P2A-GS(x2)-HRV-3C-GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTAGCAAGGCTCTGCTGAAAGGAGTGCGGGATTTCAACCCCATCAGCGCATGCGTG TGCCTGCTGGAGAACAGCTCTGACGGACACTCTGAAAGACTCTTCGGCATCGGCTTCGGCCCTTACATCATC GCCAATCAGCACCTTTTCCGGAGAAACAACGGCGAGCTGACAATCAAGACCATGCACGGAGAGTTCAAGGTG AAGAATAGTACACAGCTGCAGATGAAACCCGTTGAGGGACGGGACATCATCGTGATCAAGATGGCTAAGGAC TTCCCTCCATTCCCCCAGAAACTGAAGTTCCGGCAGCCTACCATCAAGGATAGAGTGTGTATGGTGAGCACC AACTTCCAGCAGAAGAGCGTATCCAGCCTGGTGTCCGAAAGCAGTCACATCGTGCATAAGGAAGACACCTCC TTCTGGCAGCACTGGATCACCACCAAGGACGGACAATGTGGCTCTCCTCTGGTGAGCATCATTGACGGCAAC3915-P1351WO.UW -73-ATCCTGGGCATCCACTCTCTCACACATACCACAAATGGGAGCAACTACTTCGTGGAGTTCCCTGAAAAGTTT GTGGCCACGTACCTGGATGCCGCTGACGGCTGGTGCAAGAATTGGAAGTTCAACGCCGATAAGATCAGCTGG GGCAGCTTCACCCTGGTGGAGGATGCCCCTGAGGATGACTTCATGGCCAAGAAGACCGTCGCCGCCATCATG GACGGTAGCGGCAGCGCCACAAACTTCAGCCTCCTCAAGCAGGCTGGGGACGTTGAGGAGAACCCTGGCCCC GGCTCCGGCTCTGGACCTAACACCGAGTTCGCCCTGAGCCTACTGAGAAAGAACATCATGACCATCACCACC AGCAAGGGCGAGTTCACCGGCCTGGGCATCCACGACAGAGTGTGCGTGATCCCCACCCATGCCCAGCCGGGA GATGATGTGTTGGTGAACGGCCAAAAAATCCGGGTGAAGGACAAGTACAAGCTGGTGGACCCTGAAAACATC AACCTGGAACTGACCGTGCTGACACTGGATCGGAATGAAAAATTTCGAGACATCAGAGGCTTTATCTCTGAG GACCTGGAAGGCGTCGACGCCACACTGGTGGTCCACAGCAACAACTTTACAAATACCATCTTGGAGGTGGGC CCTGTGACCATGGCTGGCCTGATCAACCTGTCTAGCACACCTACAAACAGAATGATCCGATACGACTACGCC ACAAAAACCGGCCAGTGTGGCGGAGTGCTGTGCGCCACTGGCAAAATTTTCGGCATTCACGTGGGAGGCAAT GGCAGACAGGGCTTCTCTGCCCAGCTGAAGAAGCAGTACTTCGTGGAAAAGCAGGGCTCCGGCAGTGCCACA AATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCGGGAGCGGCTCCGTGAGCAAG GGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAAGGCAGCGTGAAC GGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGACCCTACGAGGGAACACAGACCGCCAAGCTGAAA GTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATCCTGAGCCCTCAGTTCATGTATGGCAGCAAGGCA TATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAACTGTCTTTTCCTGAGGGATTCAAGTGGGAGCGT GTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACCCAGGACAGCTCCCTCCAGGACGGCGAGTTCATC TACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGACGGCCCCGTGATGCAGAAGAAAACAATGGGCTGG GAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCCCTGAAAGGCGAAATCAAACAAAGACTGAAGCTG AAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACCTACAAGGCCAAAAAGCCTGTGCAGCTGCCTGGC GCCTACAACGTGAATATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAGCAGTACGAG CGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAGCTGTACAAGTGA (SEQ ID NO:35) V and T Proteases & mCherry MGSS- TVMV-GS(x2)-P2A-GS(x2)-SuperTEV-GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTAGCAAGGCTCTGCTGAAAGGAGTGCGGGATTTCAACCCCATCAGCGCATGCGTG TGCCTGCTGGAGAACAGCTCTGACGGACACTCTGAAAGACTCTTCGGCATCGGCTTCGGCCCTTACATCATC GCCAATCAGCACCTTTTCCGGAGAAACAACGGCGAGCTGACAATCAAGACCATGCACGGAGAGTTCAAGGTG AAGAATAGTACACAGCTGCAGATGAAACCCGTTGAGGGACGGGACATCATCGTGATCAAGATGGCTAAGGAC TTCCCTCCATTCCCCCAGAAACTGAAGTTCCGGCAGCCTACCATCAAGGATAGAGTGTGTATGGTGAGCACC AACTTCCAGCAGAAGAGCGTATCCAGCCTGGTGTCCGAAAGCAGTCACATCGTGCATAAGGAAGACACCTCC TTCTGGCAGCACTGGATCACCACCAAGGACGGACAATGTGGCTCTCCTCTGGTGAGCATCATTGACGGCAAC3915-P1351WO.UW -74-ATCCTGGGCATCCACTCTCTCACACATACCACAAATGGGAGCAACTACTTCGTGGAGTTCCCTGAAAAGTTT GTGGCCACGTACCTGGATGCCGCTGACGGCTGGTGCAAGAATTGGAAGTTCAACGCCGATAAGATCAGCTGG GGCAGCTTCACCCTGGTGGAGGATGCCCCTGAGGATGACTTCATGGCCAAGAAGACCGTCGCCGCCATCATG GACGGTAGCGGCAGCGCCACAAACTTCAGCCTCCTCAAGCAGGCTGGGGACGTTGAGGAGAACCCTGGCCCC GGCTCCGGCTCTGGGGAGAGCCTGTTTAAAGGCCCCAGAGATTATAATCCCATCAGCTCCACCATCGTGCAC CTGACAAACGAGAGCGACGGACACACCACAAGCCTGTACGGAATCGGCTTTGGACCTTTCATCATTACCAAC AAGCACCTGTTCAGACGGAACAACGGCACGCTGGTGGTGCAGAGCCTGCACGGCGTTTTTAAGGTGAAGAAC ACCACCACCCTGCAGCAGCATCTGATCGACGGAAGAGACATGATCATCATCAGGATGCCTAAGGACTTCCCA CCTTTCCCTCAGAAGCTGAAATTCAGGGAGCCTCAGAGAGAGGAGCGGATCGTCCTGGTGACCACCAACTTC CAGACAAAAAGCATGAGCTCTATGGTGTCCGATACAAGCAGCACCTTCCCTTCCGGCGACGGCATCTTCTGG AAGCACTGGATCCAAACCAAAGATGGCCAGTGCGGCTCTCCCCTGGTGAGCACTAGAGATGGCTTCATCGTG GGCATCCACAGCGCTAGCAACTTCACCAACACTAACAACTACTTTACCAGCGTTCCTAAGAATTTCATGGAA CTGCTGACCAACCAGGAAGCCCAACAATGGGTGAGCGGTTGGCGGCTGAACGCCGATTCTGTGCTGTGGGGA GGACACAAGGTATTCATGGATAAGCCTGAAGAACCTTTCCAGCCTGTCAAAGAGGCCACACAGCTGATGAAC CGGAGAAGAAGAAGAGGCAGCGGCAGCGCCACCAATTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAA AACCCAGGCCCAGGTAGCGGCTCGGTGAGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATG AGATTCAAGGTGCACATGGAAGGCAGCGTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGA CCCTACGAGGGAACACAGACCGCCAAGCTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATC CTGAGCCCTCAGTTCATGTATGGCAGCAAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAA CTGTCTTTTCCTGAGGGATTCAAGTGGGAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACC CAGGACAGCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGAC GGCCCCGTGATGCAGAAGAAAACAATGGGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCC CTGAAAGGCGAAATCAAACAAAGACTGAAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACC TACAAGGCCAAAAAGCCTGTGCAGCTGCCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACCTCCCAC AACGAGGACTACACCATCGTGGAGCAGTACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAG CTGTACAAGTGA (SEQ ID NO:36) V and T and C Proteases & mCherry MGSS-TVMV-GS(x2)-P2A-GS(x2)- SuperTEV-GS(x2)-P2A-GS(x2)-HRV-3C- GS(x2)-P2A-GS(x2)-mCherry* ATGGGCTCCTCTAGCAAGGCTCTGCTGAAAGGAGTGCGGGATTTCAACCCCATCAGCGCATGCGTG TGCCTGCTGGAGAACAGCTCTGACGGACACTCTGAAAGACTCTTCGGCATCGGCTTCGGCCCTTACATCATC GCCAATCAGCACCTTTTCCGGAGAAACAACGGCGAGCTGACAATCAAGACCATGCACGGAGAGTTCAAGGTG3915-P1351WO.UW -75-AAGAATAGTACACAGCTGCAGATGAAACCCGTTGAGGGACGGGACATCATCGTGATCAAGATGGCTAAGGAC TTCCCTCCATTCCCCCAGAAACTGAAGTTCCGGCAGCCTACCATCAAGGATAGAGTGTGTATGGTGAGCACC AACTTCCAGCAGAAGAGCGTATCCAGCCTGGTGTCCGAAAGCAGTCACATCGTGCATAAGGAAGACACCTCC TTCTGGCAGCACTGGATCACCACCAAGGACGGACAATGTGGCTCTCCTCTGGTGAGCATCATTGACGGCAAC ATCCTGGGCATCCACTCTCTCACACATACCACAAATGGGAGCAACTACTTCGTGGAGTTCCCTGAAAAGTTT GTGGCCACGTACCTGGATGCCGCTGACGGCTGGTGCAAGAATTGGAAGTTCAACGCCGATAAGATCAGCTGG GGCAGCTTCACCCTGGTGGAGGATGCCCCTGAGGATGACTTCATGGCCAAGAAGACCGTCGCCGCCATCATG GACGGTAGCGGCAGCGCCACAAACTTCAGCCTCCTCAAGCAGGCTGGGGACGTTGAGGAGAACCCTGGCCCC GGCTCCGGCTCTGGGGAGAGCCTGTTTAAAGGCCCCAGAGATTATAATCCCATCAGCTCCACCATCGTGCAC CTGACAAACGAGAGCGACGGACACACCACAAGCCTGTACGGAATCGGCTTTGGACCTTTCATCATTACCAAC AAGCACCTGTTCAGACGGAACAACGGCACGCTGGTGGTGCAGAGCCTGCACGGCGTTTTTAAGGTGAAGAAC ACCACCACCCTGCAGCAGCATCTGATCGACGGAAGAGACATGATCATCATCAGGATGCCTAAGGACTTCCCA CCTTTCCCTCAGAAGCTGAAATTCAGGGAGCCTCAGAGAGAGGAGCGGATCGTCCTGGTGACCACCAACTTC CAGACAAAAAGCATGAGCTCTATGGTGTCCGATACAAGCAGCACCTTCCCTTCCGGCGACGGCATCTTCTGG AAGCACTGGATCCAAACCAAAGATGGCCAGTGCGGCTCTCCCCTGGTGAGCACTAGAGATGGCTTCATCGTG GGCATCCACAGCGCTAGCAACTTCACCAACACTAACAACTACTTTACCAGCGTTCCTAAGAATTTCATGGAA CTGCTGACCAACCAGGAAGCCCAACAATGGGTGAGCGGTTGGCGGCTGAACGCCGATTCTGTGCTGTGGGGA GGACACAAGGTATTCATGGATAAGCCTGAAGAACCTTTCCAGCCTGTCAAAGAGGCCACACAGCTGATGAAC CGGAGAAGAAGAAGAGGCAGCGGCAGCGCCACCAATTTCAGCCTGCTGAAGCAGGCTGGCGACGTGGAGGAA AACCCAGGCCCAGGTAGCGGCTCGGGACCTAACACCGAGTTCGCCCTGAGCCTACTGAGAAAGAACATCATG ACCATCACCACCAGCAAGGGCGAGTTCACCGGCCTGGGCATCCACGACAGAGTGTGCGTGATCCCCACCCAT GCCCAGCCGGGAGATGATGTGTTGGTGAACGGCCAAAAAATCCGGGTGAAGGACAAGTACAAGCTGGTGGAC CCTGAAAACATCAACCTGGAACTGACCGTGCTGACACTGGATCGGAATGAAAAATTTCGAGACATCAGAGGC TTTATCTCTGAGGACCTGGAAGGCGTCGACGCCACACTGGTGGTCCACAGCAACAACTTTACAAATACCATC TTGGAGGTGGGCCCTGTGACCATGGCTGGCCTGATCAACCTGTCTAGCACACCTACAAACAGAATGATCCGA TACGACTACGCCACAAAAACCGGCCAGTGTGGCGGAGTGCTGTGCGCCACTGGCAAAATTTTCGGCATTCAC GTGGGAGGCAATGGCAGACAGGGCTTCTCTGCCCAGCTGAAGAAGCAGTACTTCGTGGAAAAGCAGGGCTCC GGCAGTGCCACAAATTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAAGAGAACCCTGGCCCCGGGAGCGGC TCCGTGAGCAAGGGCGAGGAAGATAACATGGCTATCATCAAGGAGTTCATGAGATTCAAGGTGCACATGGAA GGCAGCGTGAACGGTCACGAGTTTGAGATCGAGGGCGAAGGCGAAGGCAGACCCTACGAGGGAACACAGACC GCCAAGCTGAAAGTGACAAAGGGCGGACCTCTGCCATTCGCCTGGGACATCCTGAGCCCTCAGTTCATGTAT GGCAGCAAGGCATATGTGAAGCACCCCGCCGATATCCCTGACTACCTGAAACTGTCTTTTCCTGAGGGATTC AAGTGGGAGCGTGTGATGAACTTCGAGGACGGCGGGGTCGTGACCGTGACCCAGGACAGCTCCCTCCAGGAC3915-P1351WO.UW -76-GGCGAGTTCATCTACAAGGTGAAGCTGAGGGGCACAAACTTCCCCTCTGACGGCCCCGTGATGCAGAAGAAA ACAATGGGCTGGGAGGCCAGCTCAGAAAGAATGTACCCTGAGGACGGAGCCCTGAAAGGCGAAATCAAACAA AGACTGAAGCTGAAGGATGGAGGCCACTACGACGCCGAAGTCAAGACAACCTACAAGGCCAAAAAGCCTGTG CAGCTGCCTGGCGCCTACAACGTGAATATCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGTG GAGCAGTACGAGCGGGCCGAGGGCAGACACAGCACCGGCGGCATGGACGAGCTGTACAAGTGA (SEQ ID NO:37) NON-LIMITING EMBODIMENTS

[0188] While general features of the disclosure are described and shown and particular features of the disclosure are set forth in the claims, the following non-limiting embodiments relate to features, and combinations of features, that are explicitly envisioned as being part of the disclosure. The following non-limiting Embodiments contain elements that are modular and can be combined with each other in any number, order, or combination to form a new non-limiting Embodiment, which can itself be further combined with other non-limiting Embodiments.

[0189] Embodiment 1. A biomaterial comprising a biological circuit configured for a conditional release of a cargo from an immobilized portion of the biomaterial, the biological circuit comprising: a polypeptide comprising a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; and wherein one or more protease sites is / are proteolytically cleaved, the conditional release occurs for release of the cargo from the biomaterial.

[0190] Embodiment 2. The biomaterial of Embodiment 1 or any other Embodiment, wherein the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0191] Embodiment 3. The biomaterial of any one of Embodiments 1-2 or any other Embodiment, wherein the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for3915-P1351WO.UW -77-the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0192] Embodiment 4. The biomaterial of any one of Embodiments 1-3 or any other Embodiment, wherein the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0193] Embodiment 5. The biomaterial of any one of Embodiments 1-4 or any other Embodiment, wherein the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0194] Embodiment 6. The biomaterial of any one of Embodiments 1-5 or any other Embodiment, wherein the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0195] Embodiment 7. The biomaterial of any one of Embodiments 1-6 or any other Embodiment, wherein the biological circuit comprises a 5-input-responsive [AND / (OR)]OR(AND) gate.

[0196] Embodiment 8. The biomaterial of Embodiment 7 or any other Embodiment, wherein the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

[0197] Embodiment 9. The biomaterial of any one of Embodiments 1-8 or any other Embodiment, wherein the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.3915-P1351WO.UW -78-

[0198] Embodiment 10. The biomaterial of Embodiment 9 or any other Embodiment, wherein the cyclic motif comprises one or more exteins formed by a split- intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.

[0199] Embodiment 11. The biomaterial of Embodiment 9 or any other Embodiment, wherein the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

[0200] Embodiment 12. The biomaterial of any one of Embodiments 1-11 or any other Embodiment, wherein the immobilized portion of the biomaterial comprises a solid support and the conditional release occurs for release of the cargo from the solid support.

[0201] Embodiment 13. The biomaterial of any one of Embodiments 1-12 or any other Embodiment, comprising a liquid matrix that comprises a biological agent configured to bind to the cargo.

[0202] Embodiment 14. The biomaterial of Embodiment 13 or any other Embodiment, wherein the biological agent is a cell or a portion thereof that comprises an antigen, and wherein the cargo is an antibody or a portion thereof that comprises an antigen- binding domain.

[0203] Embodiment 15. A biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising the biomaterial of any one of Embodiments 1-15 or any other Embodiment.

[0204] Embodiment 16. A polypeptide, comprising: a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; and a cargo, wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; wherein one or more protease sites is / are proteolytically cleaved, a conditional release occurs for release of the cargo from the polypeptide.

[0205] Embodiment 17. The polypeptide of Embodiment 16 or any other Embodiment, wherein the conditional release comprises an AND gate that requires3915-P1351WO.UW -79-proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0206] Embodiment 18. The polypeptide of any one of Embodiments 16-17 or any other Embodiment, wherein the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

[0207] Embodiment 19. The polypeptide of any one of Embodiments 16-18 or any other Embodiment, wherein the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

[0208] Embodiment 20. The polypeptide of any one of Embodiments 16-19 or any other Embodiment, wherein the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

[0209] Embodiment 21. The polypeptide of any one of Embodiments 16-20 or any other Embodiment, wherein the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

[0210] Embodiment 22. The polypeptide of any one of Embodiments 16-21 or any other Embodiment, wherein the biological circuit comprises a 5-input-responsive [AND / (OR)]OR(AND) gate.

[0211] Embodiment 23. The polypeptide of Embodiment 22 or any other Embodiment, wherein the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V3915-P1351WO.UW -80-(ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

[0212] Embodiment 24. The polypeptide of any one of Embodiments 16-23 or any other Embodiment, wherein the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

[0213] Embodiment 25. The polypeptide of Embodiment 24 or any other Embodiment, wherein the cyclic motif comprises one or more exteins formed by a split- intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.

[0214] Embodiment 26. The polypeptide of Embodiment 24 or any other Embodiment, wherein the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

[0215] Embodiment 27. A nucleic acid comprising a polynucleotide sequence encoding the polypeptide of any one of Embodiments 16-26 or any other Embodiment.

[0216] Embodiment 28. The nucleic acid of Embodiment 27 or any other Embodiment, wherein the polynucleotide sequence comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identity with at least one sequence, or at least one portion of a sequence, selected from the group consisting of: SEQ ID NOs:1-37.

[0217] Embodiment 29. An expression cassette and / or expression vector comprising the polynucleotide sequence of any one of Embodiments 27-28 or any other Embodiment configured for expression of the polypeptide in a biological system.

[0218] Embodiment 30. A genetically modified host cell comprising the nucleic acid of any one of Embodiments 27-28 or any other Embodiment.

[0219] Embodiment 31. A host organism comprising the genetically modified host cell of Embodiment 30 or any other Embodiment.

[0220] Embodiment 32. A biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising the genetically modified host cell of Embodiment 30 or any other Embodiment.3915-P1351WO.UW -81-

[0221] While illustrative embodiments have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.3915-P1351WO.UW -82-

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A biomaterial comprising a biological circuit configured for a conditional release of a cargo from an immobilized portion of the biomaterial, the biological circuit comprising: a polypeptide comprising a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; and wherein one or more protease sites is / are proteolytically cleaved, the conditional release occurs for release of the cargo from the biomaterial.

2. The biomaterial of claim 1, wherein the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

3. The biomaterial of claim 1, wherein the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

4. The biomaterial of claim 1, wherein the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.3915-P1351WO.UW -83-5. The biomaterial of claim 1, wherein the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

6. The biomaterial of claim 1, wherein the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

7. The biomaterial of claim 1, wherein the biological circuit comprises a 5- input-responsive [AND / (OR)]OR(AND) gate.

8. The biomaterial of claim 7, wherein the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide; wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

9. The biomaterial of claim 1, wherein the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

10. The biomaterial of claim 9, wherein the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.3915-P1351WO.UW -84-11. The biomaterial of claim 9, wherein the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

12. The biomaterial of claim 1, wherein the immobilized portion of the biomaterial comprises a solid support and the conditional release occurs for release of the cargo from the solid support.

13. The biomaterial of claim 1, comprising a liquid matrix that comprises a biological agent configured to bind to the cargo.

14. The biomaterial of claim 13, wherein the biological agent is a cell or a portion thereof that comprises an antigen, and wherein the cargo is an antibody or a portion thereof that comprises an antigen-binding domain.

15. A biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising the biomaterial of claim 1.

16. A polypeptide, comprising: a first protease site configured to be proteolytically cleaved for conditional dissociation of a first portion of the polypeptide from a second portion of the polypeptide, and a second protease site configured to be proteolytically cleaved for conditional dissociation of a third portion of the polypeptide from a fourth portion of the polypeptide; and a cargo, wherein the cargo is directly or indirectly linked to the first portion, the second portion, the third portion, the fourth portion, or any combination thereof; wherein one or more protease sites is / are proteolytically cleaved, a conditional release occurs for release of the cargo from the polypeptide.

17. The polypeptide of claim 16, wherein the conditional release comprises an AND gate that requires proteolytic cleavage of both the first protease site and the second3915-P1351WO.UW -85-protease site for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

18. The polypeptide of claim 16, wherein the conditional release comprises an OR gate that requires proteolytic cleavage of the first protease site or the second protease site, but not both, for the conditional release to occur for release of the cargo from the immobilized portion of the biomaterial.

19. The polypeptide of claim 16, wherein the biological circuit comprises: an AND gate that requires proteolytic cleavage of two YES gates for the AND gate to occur for release of the cargo from the immobilized portion of the biomaterial; and an OR gate that requires proteolytic cleavage of either one of two YES gates, but not both, for the OR gate to occur for release of the cargo from the immobilized portion of the biomaterial.

20. The polypeptide of claim 16, wherein the first and second protease sites are YES gates independently selected from the group consisting of: A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP); wherein A is proteolytically cleavable by eSrtA(2A9), S is proteolytically cleavable by eSrtA(4S9), T is proteolytically cleavable by an evolved potyviral TEV protease, V is proteolytically cleavable by potyviral Tobacco Vein Mottling Virus (TVMV) protease, and C is proteolytically cleavable by human rhinovirus-3C (HRV-3C) protease.

21. The polypeptide of claim 16, wherein the conditional release comprises one or more combination OR / (AND) gates, one or more combination AND / (OR) gates, one or more combination (OR / OR) gates, one or more combination (AND / AND) gates, or any combination thereof.

22. The polypeptide of claim 16, wherein the biological circuit comprises a 5- input-responsive [AND / (OR)]OR(AND) gate.

23. The polypeptide of claim 22, wherein the 5-input-responsive [AND / (OR)]OR(AND) gate comprises a [(A∨S)∧T]∨(C∧V) polypeptide;3915-P1351WO.UW -86-wherein A (LAET↓G), S (LPES↓G), T (ENLYFQ↓S), V (ETVRFQ↓S), and C (LEVLFQ↓GP) are proteolytically cleavable by eSrtA(2A9), eSrtA(4S9), an evolved potyviral TEV protease, potyviral Tobacco Vein Mottling Virus (TVMV) protease, and human rhinovirus-3C (HRV-3C) protease, respectively.

24. The polypeptide of claim 16, wherein the polypeptide comprises a cyclic motif, a tadpole motif, an n-armed star motif, an H-shape motif, or any combination thereof.

25. The polypeptide of claim 24, wherein the cyclic motif comprises one or more exteins formed by a split-intein circular ligation of peptides and proteins (SICLOPPS) technique that produces, as one or more byproducts of the SICLOPPS technique, one or more inteins.

26. The polypeptide of claim 24, wherein the tadpole motif, the n-armed star motif, and / or the H-shape motif is / are formed by at least one ligation technique selected from the group consisting of: SpyLigation, SnoopLigation, and DogLigation.

27. A nucleic acid comprising a polynucleotide sequence encoding the polypeptide of claim 16.

28. The nucleic acid of claim 27, wherein the polynucleotide sequence comprises at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% identity with at least one sequence, or at least one portion of a sequence, selected from the group consisting of: SEQ ID NOs:1-37.

29. An expression cassette and / or expression vector comprising the polynucleotide sequence of claim 27 configured for expression of the polypeptide in a biological system.

30. A genetically modified host cell comprising the nucleic acid of claim 27.

31. A host organism comprising the genetically modified host cell of claim 30.3915-P1351WO.UW -87-32. A biosensor, implantable biosensor, medical device, implantable medical device, drug delivery device, or implantable drug delivery device comprising the genetically modified host cell of claim 30.3915-P1351WO.UW -88-

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