WO2018129021A1 - Modular, inducible repressors for the control of gene expression - Google Patents

Abstract

A modular, inducible repressor for control of gene expression is disclosed. Generally, the repressor comprises a DNA binding protein fused to a protein that binds to an exogenous inducer molecule, wherein the repressor is directly inducible by the exogenous inducer molecule. In particular embodiments, the DNA binding protein comprises a transcription activator-like effector (TALE) and the protein that binds to an exogenous inducer molecule comprises glycine betaine binding protein (GBBP). In such embodiments, the exogenous inducer molecule comprises glycine betaine.

Description

MODULAR, INDUCIBLE REPRESSORS FOR THE

CONTROL OF GENE EXPRESSION

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No.

62/443,376, filed on January 6, 2017, which is hereby incorporated by reference for all purposes as if fully set forth herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED

ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 2, 2018, is named P14178-02_ST25.txt and is 80,522 bytes in size.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant numbers CBET-1402101 and CBET-1149678, awarded by the National Science Foundation and ROl GM066972, awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Progress in genetic engineering, metabolic engineering, and synthetic biology will be accelerated by the development of versatile, user-friendly technologies to manipulate cells with precision and efficiency. Recognition of an external stimulus (input) resulting in a particular cellular response (output) is essential to create engineered cells with therapeutic functions or for their use as efficient bio-factories. The ability to respond to an input is often closely linked to the fine control of gene expression. A key strategy for optimizing this control is the design and rearrangement of regulatory elements for gene expression. Although many tools have been developed, most of these act in cis (e.g., promoters, ribosome binding sites and terminators), and are limited by compatibility, ease of implementation, difficulty to optimize gene expression under multiple conditions, and undesirable effects in the native physiology of the microorganism (Copeland, et al, 2014).

Natural inducible repressors, such as Lacl and TetR, have been broadly used as important elements to control recombinant gene expression, as well as to build complex genetic circuits (Cameron, et al, 2014). These repressors, however, have specificity to unique DNA sequences (i.e., specific operators), and this single DNA sequence-dependency limits their flexibility. Moreover, repressors, such as Lacl and TetR, use the expensive inducers isopropyl β-D-l-thiogalactopyranoside (IPTG) and anhydrotetracycline, respectively, which makes the use of these natural repressors economically unfeasible on an industrial scale.

One approach to engineering new conditional repressors is to use directed evolution or rational design to modify the effector specificity of natural conditional repressors. Directed evolution using random or saturation mutagenesis led to versions of TetR (Scholz, et al, 2003), AraC (Tang, et al, 2008; Tang, et al, 2013) and XylR (Galvao, et al, 2007) with altered effector specificity. A combination of

computational design and directed evolution led to versions of Lacl that respond to new ligands with comparable specificity and induction levels (Taylor, et al, 2016; Meinhardt, et. al., 2012). Due to the nature of the DNA binding properties of these proteins, however, they cannot be readily redesigned to control promoters that lack their cognate binding sites (i.e., they are not modular). The DNA binding proteins zinc fingers (ZF), transcription activator-like effectors (TALE) and the Cas9 protein from the clustered regularly interspaced short palindromic repeats (CRISPR) system are proteins that, to different degrees, can readily be programmed to specifically interact with any desired target DNA sequence(Copeland, et al, 2014; Kabadi, et al, 2014; Wright, et al, 2006).

Activators are created by fusing ZF/TALE/Cas9 proteins to transcriptional activator domains, whereas repressors typically rely on the ZF/TALE/Cas9 proteins directly blocking transcription. With the exception of a light inducible system for recruiting transcriptional activators to associate with TALE proteins (Konermann, et al, 2013), the drawback of such activator or repressor proteins is they are not directly inducible by exogenous molecules/signals. They must rely on existing inducible repressor systems for controlling their expression or controlling the expression of a protease designed to degrade repressors engineered with protease sites (Copeland, et al, 2016). Such systems do not provide new, reversibly modulatable proteins or novel inducer molecules.

A potential strategy for creating inducible repressors is the use of domain insertion to fuse a ZF/TALE/Cas9 protein and a protein that binds a desired inducer molecule such that the inducer molecule reduces the ZF/TALE/Cas9 domain's affinity for DNA (Ostermeier, 2005). Domain insertion has been shown to be useful for creating switch proteins in general (Stein, et al, 2015). Such switches function by two non-exclusive mechanisms (Tucker, et al, 2001). Ligand-binding can modulate the specific activity of the fusion protein, as happens in natural heterotropic allosteric proteins (e.g., Lacl), or ligand-binding can thermodynamically/proteolytically stabilize the fusion such that it accumulates at higher levels in the cell (Heins, et al, 2011 ; Choi, et al, 2013; Banaszynski, et al, 2006). The latter mechanism presumably could only be used to create artificial transcription factors in which ligand-binding causes increased binding to DNA, such as fusions for which ligand-binding causes repression (i.e., a conditional repressor) (Oakes, et al, 2016) or causes activation (i.e., a conditional activator) (Feng, et al, 2015). By contrast, an inducible repressor (i.e., the inducer causes derepression) presumably could not function by this mechanism and would require instead the allosteric mechanism. A recent study of fusions of a ZF and maltose binding protein suggests that domain insertion can be used to create inducible repressors, but the dynamic range achieved was small (a 2- to 3-fold expression increase in the presence of maltose) (Younger, et al, 2016). Whether domain insertion can be used to build conditional repressors that rival Lacl or TetR is uncertain.

SUMMARY

Generally, the presently disclosed subject matter provides a modular, inducible repressor for control of gene expression, the repressor comprising a DNA binding protein fused to a protein that binds to an exogenous inducer molecule;

wherein the repressor is directly inducible by the exogenous inducer molecule.

More particularly, in some aspects, the presently disclosed subject matter uses domain insertion and directed evolution to convert a TALE into a modular, single polypeptide chain inducible repressor with a large dynamic range whose ability to repress can be alleviated by the inexpensive osmolyte glycine betaine (GB). Accordingly, in some aspects, the DNA binding protein comprises a transcription activator-like effector (TALE). Likewise, in some aspects, the protein that binds to an exogenous inducer molecule comprises glycine betaine binding protein (GBBP). In such aspects, the exogenous inducer molecule comprises glycine betaine.

In particular aspects, presently disclosed modular, inducible repressor comprises a betaine-inducible transcriptional factor (BITE). In certain embodiments, the betaine-inducible transcriptional factor (BITE) is selected from a protein having a sequence at least 90%, 95%, or 100% identical to any one of BITElacO-04,

BITElacO-14, BITElacO-22, BITElacO-H7, BITElacO-C7, BITElacO-GlO, BITElacO-D2, and BITElysA.

In some aspects, the presently disclosed subject matter provides a method of controlling gene expression in a cell, the method comprising contacting the cell with a presently disclosed modular, inducible repressor.

In other aspects, the presently disclosed subject matter provides a biosensor comprising a presently disclosed modular, inducible repressor.

Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

FIG. 1 is a structural representation of the protein TALE. Each DNA-binding module has 32 highly conserved residues and two hypervariable residues (RVDs) in the 12th and 13th amino acid positions of each repeat. The RVDs specify the DNA base being targeted according to the code NG = T, HD = C, NI = A, and NN = G or A. Flanking the binding domain are the N-ter domain and the C-terminal domain carrying the translocation, NLS: Nuclear Localization Signal and AD: Activator Domain. Prior art figure from Sanjana et al. (2012); FIGS. 2 A and 2B show (A) scheme of the heterodimerization. Light induces dimerization of CRY2 and CIB1, recruiting the effector to the target promoter Konermann et al. (2013) (prior art); (B) Scheme of system mediated by proteolysis (Copeland et al. (2016) (prior art). TALE contain tev protease cleavage sites binds to target promoter inhibiting its expression. Gene expression occurs by proteolytic cleavage of the TALE in consequence of induction of expression of the tev protease;

FIGS. 3 A, 3B, 3C, and 3D show construction and selection of the BITE system: (A) Schematic of the function of BITE, an allostatic inducible repressor. BITE is derived from the insertion of a circular permuted glycine betaine binding protein (cpGBBP) into a TALE protein. The TALE domain of BITE is designed to bind DNA at a TALE binding site (TBS) located between the promoter and the ribosome binding site (RBS). BITE binding represses transcription of the target gene. The DNA affinity of BITE is reduced by the binding of glycine betaine (GB) to the cpGBBP domain, resulting in induction of gene expression; (B) selection system for identifying BITE proteins from a combinatorial library of cpGBBP-TALE fusions. Plasmids pTSl and pDIM-C8 are compatible and essential components of the bandpass filter system. In the absence of sufficient cellular β-lactamase (BLA) activity for hydrolysis of ampicillin (Amp), cell wall synthesis is compromised and cells cannot proliferate. In addition, cell wall breakdown results in the accumulation of aM- pentapeptide (aM-Pp), which induces the ampC promoter via interactions with AmpR, resulting in the production of TetC, which confers tetracycline (Tet) resistance. The level of Amp necessary to induce ampC is lower than the level that prevents the growth of E. coli cells. Thus, cells that hydrolyze Amp too efficiently cannot grow in the presence of Tet. pTSl also contains the library of different cpGBBP inserted into different locations of TALE; (C) demonstration of the band-pass system and

TALElacOl repression. As a result of the band-pass genetic circuit and the addition of Tet to the growth medium, cells expressing BLA without repression only grow at high concentrations of Amp (left). When TALElacOl is co-expressed to repress the expression of BLA, growth in the presence of Tet only occurs at low Amp

concentrations (right); and (D) Library selection. Members of the library of cpGBBP- TALE fusions will have different abilities to repress BLA expression. The negative selection is applied by recovering the colonies from plates lacking GB and containing Tet and low Amp concentrations. These variants are subjected to the second, positive selection step in which cells must grow at high Amp in the presence of GB. The two- tiered selection identifies BITE inducible repressors that cause low Amp resistance is the absence of GB and high Amp resistance in the presence of GB;

FIGS. 4A, 4B, 4C, and 4D show the characterization of BITElacO-C7 controlling expression from /ac-derived promoters on plasmids and the chromosome: (A) Scheme of the three different reporter systems used to assess BITElacO-C7's abilities as a GB-inducible repressor; (B) GB-dependence of the minimum inhibitory concentrations (MIC) for ampicillin for cells in which expression of beta-lactamase from the tac promoter is regulated by Lacl, TALElacO, or BITElacO-C7. The fold- increase in the MIC by the inclusion of 10 mM GB in the media is indicated; (C) GB- dependence of the mean sfGFP protein production rates for cells expressing Lacl, TALELacOlor BITElacO-C7 targeting a plasmid-borne sfGFP under the control of the tac promoter; and (D) GB-dependence of the mean sfGFP protein production rates cells expressing Lacl, TALELacOlor BITElacO-C7 targeting a chromosomal sfGFP reporter integrated into the lacIZYA locus. The error bars represent the standard deviation (n=3);

FIGS. 5 A and 5B show the characterization of BITElacO-C7 controlling constitutive promoters with single or multiple TBS. (A) GB-dependence of the mean sfGFP protein production rates for cells expressing BITElacO-C7 targeting a plasmid- borne sfGFP reporter under control of the P100 or P106 promoters with a single TBS. The control data is from cells not expressing BITElacO-C7. The error bars represent the standard deviation (n=3); and (B) GB-dependence of the MIC for ampicillin for cells in which BITElacO-C7 is regulating expression of beta-lactamase under control of the P100 or P106 promoters with single or multiple TBS. Black, 10 mM GB; grey, 0 mM GB. The values indicate the median MIC from three independent experiments;

FIGS. 6 A and 6B show the characterization of BITE controlling constitutive promoters with single or multiple TALE binding sites (TBS). (A) GB-dependence of the mean sfGFP protein production rates for cells expressing BITElacO-C7 targeting a plasmid-borne sfGFp reporter under control of the PI 00 or PI 06 promoters with a single TBS (n = 3). Control cells lacked BITElacO-C7; (B) GB-dependence of the MIC for ampicillin for cells in which BITElacO-C7 is regulating expression of beta- lactamase under the control of the P¹⁰⁰ or P¹⁰⁶ promoters with single or multiple TBS. The fold-increase in the MIC by the inclusion of 4 mM GB in the media is indicated. Error bars represent standard deviation; FIGS. 7 A and 7B show the modularity of BITE inducible repressors: (A) The TALELacOl repeat motifs of BITElacO-C7 were replaced by TALElysA repeat motifs to create BITElysA targeting the native E. coli lysA promoter (Plys); and (B) GB-dependence of growth of cells expressing TALElysA or BITElysA in minimal medium lacking or containing 0.4 mM lysine. LysA is essential for growth in minimal media lacking lysine. The error bars represent the standard deviation (n = 3);

FIG. 8 shows GB induction of β-lactamase expression under control of P¹⁰⁶ with one (square) or four (circle) TBS. β-lactamase induction was observed by measuring cell growth by optical density (OC) in the presence of 256 μg/mL ampicillin with increasing concentrations of GB. Error bars indicate standard deviation (n = 3);

FIGS. 9A and 9B show the validation of the selection system: (A)

TALELacOl functions as a repressor in the band-pass system. All cells contain bla under the tac promoter on pDIM-C8-BLA. Cells also harbored pTSl from which either Lacl, TALElacOl, or neither of the two proteins were constitutively expressed. Two of culture (~2 x 10⁴ cells) were spotted onto plates containing the presence or absence of 200 μg/mL Amp and 1 mM IPTG as indicated; and (B) TALE's ability to repress expression in band-pass system is not affected by GB. Cells harboring pDIM- C8-BLA and pTSl-TALElacO plasmids were plated in the presence and absence of GB (5 mM);

FIG. 10 shows the schematic representation of the library construction by semi-rational insertion of circular permuted GBBP into TALE. The construct pUC/GBBPlkGBBP containing an end-to-end fusion of the GBBP spanned by a linker GSGG was used as template to create cpGBBPs permuted in 137 position. This library of circular permutations was inserted into the linearized pTSl-TALE plasmid at the 194 predetermined positions;

FIGS. 11A, 11B, and 11C show fusion sites of TALE/cpGBBP chimeras that behave as BITE inducible repressors: (A) Structural representation of TALE showing the insertion sites of the cpGBBP in the library (red) and in BITE proteins (arrows). Insertions in the N-terminal region (1-137) are shown in blue; (B) a linear diagram of TALE protein with arrows indicating the cpGBBP insertion sites in BITE; and (C) Structural representation of GBBP showing sites of circular permutation in the library (green) and in BITE proteins (arrows). The original N- and C-termini are indicated. GB is shown in purple; FIG. 12 shows a ribbon representation of the structural model of BITElac-C7. Regions derived from GBBP are shown in red, TALE repeat domain is shown in purple and conserved N-terminal region of TALE is shown in blue;

FIGS. 13 A, 13B, and 13C show dose-response for BITElacO-C7 and

BITElacO-C7mut: (A) effect of GB concentration on the growth of cells in media containing 256 μg/mL Amp in which BITElacO-C7 regulates β-lactamase expression from the tac promoter; (B) effect of GB concentration on the relative fluorescence of cells in which BITElacO-C7 (black) or BITElacO-C7mut (gray) regulates chromosomal sfGFP expression from the tac promoter. Values are normalized to the fluorescence of cells lacking the sfGFP gene on the plasmid; and (C) effect of GB concentration on the growth of the cells in media containing 256 μg/mL Amp in which BITElacO-C7 regulates β-lactamase expression from the PI 06 promoter with single (black) or four TBS (gray). Error bars represent the standard deviation (n=3);

FIGS. 14A and 14B show characterization of BITElacO-C7 controlling expression from /ac-derived and constitutive promoters: (A) GB-dependence of the mean mCherry protein production rates for cells expressing Lacl, TALELacOlor BITElacO-C7 targeting a plasmid-borne mCherry under the control of the tac promoter (n = 3); and (B) GB-dependence of the mean eGFP protein production rates for cells expressing BITElacO-C7 targeting a plasmid-borne sfGFp reporter under control of the P100 or P106 promoters with a single TBS (n = 3); and

FIGS. 15A, 15B, 15C, and 15D show the dependence of GB-inducible BITE gene activation on the number of TBS. The graphs indicate the effect of GB on the viability of cells in media containing the indicated concentrations of Amp. In these cells, BITElacO-C7 regulates β-lactamase expression from the PI 06 promoter containing (A) one, (B) two, (C) three, and (D) four TBS. The colony forming units (CFU) was normalized to the condition with the highest CFU. Black bars, 5 mM GB; grey bars, 0 mM GB. The error bars represent the standard deviation (n = 3).

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the presently disclosed subject matter are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter set forth herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated Figures. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

I. MODULAR, INDUCIBLE REPRESSORS FOR THE CONTROL OF GENE EXPRESSION

The development of modulated, designable and customizable trans-acting regulatory tools is an important goal in the fields of synthetic biology and metabolic engineering. Such technologies are essential for the optimization of gene expression, metabolic flux, and synthetic gene networks. The presently disclosed subject matter, in some embodiments, provides modular, inducible repressors for the control of gene expression. In particular embodiments, expression is induced by the inclusion of an inexpensive, non-metabolizable compound to the culture media. The presently disclosed process provides for an efficient modulation of gene expression and can be a useful tool for a variety of biotechnological and biomedical applications. Thus, the presently disclosed modular, inducible repressors can be used as tools to control gene expression (new vectors), biosensors, and gene therapy tools.

To this end, recent advances in genetic engineering have provided a set of new tools to design organisms capable of recognizing an external stimulus (input) and producing a particular cellular response (output). Such behavior is essential to create in these organisms therapeutic functions or for them work as efficient bio-factories. The ability to respond to an input is closely linked to the fine control of gene expression in the cells. A key point to optimize this control is the

design/rearrangement of regulatory elements of gene expression. Although many tools have been developed in the last several years, most of these acts in cis, such as promoters, ribosome binding sites and terminators, and are limited by compatibility, ease of implementation, difficulty to optimize gene expression under multiple conditions and undesirable effects in the native physiology of the microorganism. Many of these problems could be solved, however, with the development trans-acting DNA-binding transcription factors that could control the expression of a target gene without changing its native regulation. Natural DNA binding proteins, such as Lacl and TetR, are well-characterized inducible repressors that have been broadly used as important elements to control recombinant protein expression, as well as to build complex genetic circuits. These repressors, however, have specificity to a unique DNA sequences (i.e., operators), and this single DNA sequence-dependency limits their flexibility. Moreover, they use expensive inducers, such as isopropyl β-D- 1-thiogalactopyranoside (IPTG) and anhydrotetracycline (aTc), which makes the use of these natural repressor economically unfeasible on an industrial scale. Thus, the development of new transcription factors capable of binding any DNA sequence has a great biotechnological relevance, especially those that could be modulated by low- cost inducers.

Transcription activator-like effectors (TALEs) are site-specific DNA-binding proteins that can be reprogramed to specifically interact with any desired DNA sequence target. New technologies based on TALE have been developed to create transcription activation or repression system. They have been generated through heterodimerization of the TALE, with transcription activation domain or by inserting protease recognition sites into the TALE backbone. The use of these approaches, however, can be limited by a small number of well-characterized ligand-dependent heterodimerization domains, low absolute level of transcriptional activation or undesired proteolysis. A single polypeptide chain allosterically controlled could overcome these limitations.

Accordingly, the presently disclosed subject matter provides an engineered TALE to create a customizable, single polypeptide chain whose ability to repress can be modulated by an inducer molecule. In some embodiments, a directed evolution approach was used for recombining the genes coding for TALE and the Escherichia coli glycine betaine binding protein (GBBP) to create a family of Betaine-Inducible Transcriptional Effector (BITE) in which glycine betaine works as an allosteric effector for TALE.

The presently disclosed subject matter demonstrates that the BITE system is able to control gene expression of gene targets, either plasmids or the chromosome. GB is not catabolized by E. coli, and it has a low cost, both of which are excellent qualities of an inducer. Therefore, the presently disclosed betaine-inducible system allows the control gene expression with efficiency and low cost, key factors for application on an industrial scale. Moreover, the presently disclosed subject matter provides an alternative approach to develop new technologies based on TALE, which expand the versatility of this protein.

TALE proteins as modular DNA binding proteins.

TAL effectors (Transcriptional Activator-Like Effectors: TALEs) are a class of structurally and functionally distinct proteins from bacteria Xanthomonas phytopathogenic. Gu et al. (2005); Boch and Bonas (2010); Morbitzer et al. (2010); and Cermak et al. (2011). This protein has been shown to be a new option for a designable DNA binding protein motif and has gained significant attention in controlling the expression of eukaryotic genes, Li et al. (2015); Zhang et al. (2011); and Mercer et al. (2014), and more recently in prokaryotes such as Escherichia coli. Politz et al. (2013); Copeland et al. (2016). Its wide applicability as a tool in synthetic biology is due the ratio of 1 : 1 between the residues of the DNA binding domain and the base pair of DNA target. Li et al. (2015); Moore et al. (2014); Garg et al. (2012).

TALE has three parts: (1) a core set of tandem repeats, this domain is responsible for binding to the target DNA sequence, (2) N-terminal region that has a signal translocation function and (3) a C-terminal region contains a transcriptional activation domain, as well as a nuclear localization signal (see FIG. 1). Deng et al. (2012).

The central DNA binding domain consists of tandem repetitions (TAL repeats), wherein each repetition recognizes a specific base pair. Each TAL repeat contains 34 highly conserved residues, with the exception of two residues at positions 12 and 13 that are hypervariable (known as RVD: Repeat Variable Di-Residues). Deng et al. (2012). These RVDs confer specificity to the target DNA. The code for recognition of DNA by the RVD was deciphered by both experimental and computational approaches. Morbitzer et al. (2010); Cermak et al. (2011); Wood et al. (2011); and Bodnar et al. (2013). The RVD containing His / Asp (HD), Asn / Gly (NG), Asn / He (NI) and Asn / Asn (NN) recognize cytosine (C), thymine (T), adenine (A) and guanine / adenine (G / A), respectively.

The binding of the TAL repetitions to DNA is modular, allowing modify the protein to bind to a sequence defined by the researcher simply modifying the RVDs. By these features, TALE can be design to bind to any promoters to control gene expression and also can be fused to a nuclease in order to edit genes in a specific way. Biffi et al. (2014); Berdien et al. (2014); Mussolino et al. (2014); Poirot et al. (2015); and Miller et al. (2011). Therefore, this protein has been applied in several fields as synthetic biology, metabolic engineering and gene therapy. Li et al. (2015); Copeland et al. (2016); Moore et al. (2014); Poirot et al. (2015); and Miller et al. (2011).

Current strategies to control gene expression using TALE

Currently, there are two main approaches to create inducible system based on TALE. Small molecule- and light-responsive systems have been generated through heterodimerization of the TALE with transcription domains. Konermann et al.

(2013). Here TALE is used to co-localize an inducible system to a desire promoter region. (FIG. 2A). However, this strategy is limited by a small number of well- characterized ligand-dependent heterodimerization domains and also by lower absolute level of transcriptional modulation as compared to single molecule transcriptional effectors.

In another strategy, TALE is designed to repress gene expression and protease recognition sites are inserted into TALE backbone (FIG. 2B). Copeland et al. (2016). Thus, TALE can be cleaved by inducing protease expression activing gene expression. The main drawbacks of this strategy are the irreversibility and the potential for undesirable proteolysis on other proteins in the cell. This system requires inducible expression of the protease and does not provide for new inducible repressors with novel inducer molecules.

Alternative approach to creating new inducible repressors

Another approach to creating new inducible repressors is illustrated by Taylor, N.D. et al. (2016). In this approach the natural E. coli lac repressor, Lacl, was engineered to respond to new inducer molecules: fucose, gentiobiose, lactitol and sucralose. However, these new repressors have specificity to a unique DNA sequences (i.e. lac operator), and this single DNA sequence-dependency limits their flexibility. Moreover, they use expensive inducers (Table 1), which makes the use of these engineered repressors economically unfeasible on an industrial scale.

Presently Disclosed Strategy

A single polypeptide chain allosterically controlled by a ligand could overcome these limitations. The presently disclosed subject matter provided, in part, the creation of an allosteric single-chain inducible repressor by the fusion of TALE with a glycine betaine binding protein (GBBP) (FIG. 3A). From a library created by random circular permutation and random insertion of GBBP in TALE, a chimeric protein was identified wherein the TALE domain's repression activity was modulated by the binding of the small molecule glycine betaine (GB) (N,N,N-trimethylglycine) to the GBBP domain. GB is not catabolized by E. coli and it has a low cost, thus making it an excellent molecule for an inducer. This fusion protein is referred to herein as a Betaine-Inducible Transcriptional Effector (BITE). BITE allows convenient control of gene expression with efficiency and low cost, key factors for application on an industrial scale. Moreover, the presently disclosed subject matter provides an alternative approach to develop new technologies based on TALE, expanding the versatility of this protein. Library creation by semirational insertion of circularly permuted GBBP (cpGBBP) into TALE

A directed evolution approach was used in which two non-homologous genes, TALE and GBBP, were recombined in vitro and subjected to selective pressure, in order to identify chimeric proteins in which the TALE domains ability to repress expression could be alleviated by the presence of GB.

Initially the GBBP gene, proX (GeneID: 947165), was amplified from i?. coli K12 genomic DNA by PCR and cloned into plasmid pUC19. The construct pUC/GBBP was the template to amplify a second copy of proX. This second fragment was cloned into the pUC/GBBP, generating the construct pUC/GBBPlkGBBP containing an end-to-end fusion of the GBBP spanned by a linker GSGG To perform the circular permutation, 140 residues that are solvent accessible were focused on, flexible, loosely packed, and between secondary structure elements, and the circularly permuted GBBPs (cpGBBPs) were generated by PCR from tandem GBBP genes.

A TALElacO gene was synthesized and codon-optimized for expression in E. coli by the Pfleger group. Politz et al. (2013). This gene was designed to bind to LacOl region of Ptac promoter. The gene was cloned in the vector pTSl under the control of a strong constitutive promoter. This construct, named pTSl-TALE, was linearized in 194 codons by multiplex inverse PCR and ligated to the circularly permuted GBBP DNA. The product of the ligation reaction was purified, concentrated and used to transform electrocompetent E. coli NEB 5-alpha (New England Biolabs, Ipswitch, MA). After growth, all the bacterial colonies present on the plates were harvested, and the library was stored at -80°C in storage media (LB + 10% glycerol (v/v)).

Selection system

Library plasmid DNA was extracted from an aliquot of cells libraries and used to transform electrocompetent E. coli SNO301 cells (ampDl, ampAl, ampC8,pyrB, recA, rpsL) harboring pDIMC8-BLA. Plasmid pDIMC8-BLA contains the TEM-1 β- lactamase (BLA) gene under the control of the Ptac promoter that TALElacO is designed to repress. The level of expression of BLA can be quantified by the minimum inhibitory concentration (MIC) for ampicillin since BLA confers resistance to ampicillin. Plasmids pTSl and pDIM-C8 are compatible and were used previously to create a genetic circuit named band-pass filter. Sohka et al. (2009). This band pass filter allows the ability to select cells that have a certain level of ampicillin resistance between and upper threshold and a lower threshold. It can thus be used to select TALElacO-GBBP fusions that repress BLA expression. To select chimeric proteins in which the TALElacO domain's repression activity was modulated by GB, this bandpass filter system was used (see details Figs. 3B, 3C, and 3D). After selection, 7 clones showed a greater than two-fold increase in MIC in the presence of GB (see Table 2, FIG. 11 and Example 4). The plasmid DNA isolated from the clone that showed the largest inducible effect (8-fold) in the presence of GB was sequenced. In this clone, the new N- and C- terminal of the GBBP were positions 130 and 131 (number according to the original GBBP protein), respectively, and this cpGBBP was inserted after the residue 136 in the TALElacO. In addition, this chimeric construct also contained two-residue linkers at each fusion site between cpGBBP and TALE (Table 1). This clone was denominated as BITElacO-C7. The other surviving clones were also sequence and characterized (Table 2 and Example 4).

BITE controlling expression on plasmids and the chromosome.

A repressor protein binds to the operator region of a promoter and blocks RNA polymerase, thereby repressing gene expression. TALElacO was designed by the Pfleger group, Politz et al. (2013), to bind to the operator LACOl of the tac promoter (Ptac). Different platforms were used to evaluate the ability of the presently disclosed BITE system to reversibly repress gene expression. For this, a two-plasmid system was constructed, consisting of a high copy number plasmid carrying the best BITE (BITElacO-C7) and a second plasmid with low copy number carrying a reporter gene. Proof-of-concept experiments were carried out in E. coli MG1655 Alacl. Two plasmidial reporters were tested: β-lactamase (bla) and mCherry. Both genes were placed under the control of the Ptac promoter on the low copy pDIM-C8 plasmid. To compare the presently disclosed BITE system with the canonical LacI-IPTG system, lad was expressed in the place of the BITElacO-C7 on the same plasmid.

The strains' level of β-lactamase expression in the presence an absence of the GB was quantified by determining the minimum inhibitory concentration of Amp (MICAmp) (FIG. 4A). As expected, little Amp resistance was observed in strains producing the repressor TALElacO either in the absence or presence of GB. However, when BITElacO-C7 was produced instead, expression was repressed in the absence of GB but the MICAmp increased 8-fold in the presence of GB (FIG. 4A). The mCherry induction was verified by measuring cell fluorescence in the presence an absence of the GB. As shown in FIG. 4B, after induction with GB the presently disclosed BITE system achieved essentially the same level of mCherry fluorescence that was observed for Lacl induced with IPTG

To probe the ability of the presently disclosed BITE system to regulate chromosomal targets, a fluorescent reporter gene (sfGFP) contain a LacOl sequence upstream was integrated into the lacIZYA locus and targeted it with BITElacO-C7. In the absence of GB, a weak sfGFP production was observed (FIG. 4C) consistent with repression of the gene. As expected, the presence of GB resulted in a significant increase of production of the sfGFP consistent with a derepression and high-level expression of sfGFP.

Modularity of BITE inducible repressors

In theory, the insertion of the GBBP domain in conserved N-ter of TALE would allow the presently disclosed BITE system to keep its modularity. To test the modularity of the presently disclosed system, the DNA-binding domain from the TALElacOl was replaced with a repeat domain targeting 22 base pairs in the lysA promoter (Plys) to create BITElysA (FIG. 7A). The lysA promoter controls the expression of the protein LysA that encodes a diaminopimelate decarboxylase that is essential for growth on minimal medium lacking lysine. It was observed that the new BITElysA protein containing the lysA targeting repeat domain was able to repress the expression of the chromosomal reporter LysA inhibiting the cells growth in absence of lysine. After 18h of induction with GB, optical density (OD) was 6-fold higher relative to the uninduced state (FIG. 7B). Therefore, the modularity of BITE is demonstrated. End users will be able to reprogram the TALE domain in the presently disclosed BITE system to repress their promoter of interest and then use GB to induce expression of their target gene. Engineering constitutive promoters to be BITE controlled

The production of commodity chemicals and biofuels are made in a lower margin than special product as pharmaceutics, and usually is not possible to use an inducible system. For this reason, many industries, generally, use constitutive promoter for protein expression. However, high unregulated heterologous expression can be toxic to the host organism and limit production. This limitation can be overcome by an efficient and inexpensive inducible system. The capacity of the presently disclosed BITE protein to control gene expression was tested using constitutive promoters. For this, a TALE binding site (TBS) was added between the gene and the constitutive promoter. Two different promoters were tested, P¹⁰⁰ and P¹⁰⁶, having the following relative strengths: 1 and 0:47 respectively.

Super-folder GFP (sfGFP) was used as a reporter gene. Under the strongest promoter ( P¹⁰⁰) BITElacO-C7 repressed sfGFP production 6-fold in relation to cells lacking the BITE gene. In the presence of GB the gene was induced 3 ± 0.2 fold (FIG. 6A). When a weaker promoter ( P¹⁰⁶) was tested, the sfGFP expression was repressed 34-fold relative to the control, and in the presence of GB sfGFP proved to be fully "on" (40 ± 18). This results suggest that the combination BITE: weak constitutive promoter leads to a greater dynamic-response window than BITE: strong constitutive promoter. The BLA gene also showed higher modulation when the gene was controlled by weaker promoter (P⁷⁰⁰: 8x and V¹⁰⁶: 16x) (FIG. 6B). This effect can be explained by leaky expression when a stronger promoter was used, decreasing the difference between "on" and "off states. Thus, to decrease the leakiness, the TBS number was increased downstream the P¹⁰⁶ constitutive promoter. As can be seen in FIG. 6B when four TBS was inserted an approximate full off state was reached, and consequentially a higher modulation was observed (64-fold). This modulation is the same repression-induction characteristic of the canonical repressor system LacI-IPTG (FIG. 4B), with the advantage of the presently disclosed TALE based system can be reprogrammed to target any DNA sequence of interest, and is modulated by a very cheap inducer.

Activation of gene expression by ligand-responsive BITE

To determine the amount of GB for full induction, how BITElacO-C7's repression depended on GB concentration using the P¹⁰⁶ controlling beta-lactamase expression was evaluated (FIG. 8). It was observed that β-lactamase expression (as measured by cell growth in the presence of 256 μg/ml ampicillin) increased in response to GB concentrations. When the gene was under the control of V¹⁰⁶ contain a single TBS the apparent ^ was 0.85 mM, and for the construction with four TBS the apparent Kd was 3.2 mM. Thus, these findings indicate that BITElacO are sufficiently sensitive for applications that require tunable forms of gene activation.

In particular embodiments, the presently disclosed subject matter provides the engineering of transcription activator-like effectors (TALEs) to function as a single polypeptide chain inducible repressor of gene expression. Transcription activator-like effectors (TALEs) are site-specific DNA-binding proteins that can be reprogramed to specifically interact with any desired DNA sequence target. Accordingly, in some embodiments of the presently disclosed subject matter, the genes coding for a TALE repressor and the Escherichia coli glycine betaine binding protein (GBBP) were recombined and directed evolution was used to create a family of Betaine-Inducible Transcriptional Effectors (BITE) for which the low-cost compound glycine betaine (GB) functions as an inducing molecule. The presently disclosed BITE system can control gene expression in plasmidial or chromosomal contexts, can be tuned through the introduction of multiple TALE binding sites, and can be redesigned to inducibly repress new promoters using the simple DNA binding design rules of TALEs. This simple and efficient modulation of gene expression achieved by the presently disclosed technology is potentially a useful tool for biotechnological applications.

More particularly, in some embodiments, the presently disclosed BITE system can be used as a biosensor. For example, in theory, GB is a disease biomarker for some metabolic syndromes, cancer and cardiovascular diseases. Since the variation of GB concentration can modulate BITE activity, the combination of BITE and a reporter gene could create a biosensor system.

Another potential application is improving gene therapy tools. For example, plasmids could be developed with BITE controlled promoters as alternatives to commonly used inducible promoters. II. GENERAL DEFINITIONS

Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this presently described subject matter belongs.

"Sequence identity" or "identity" in the context of proteins or polypeptides refers to the amino acid residues in two amino acid sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, "percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the amino acid sequence in the comparison window may comprise additions or deletions (i.e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. Useful examples of percent sequence identities include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 50% to 100%. These identities can be determined using any of the programs described herein.

Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the Meg Align™ program of the

LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized. The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp (1989) CABIOS 5: 151-153; Higgins et al. (1992) Comput. Appl. Biosci. 8: 189- 191) and found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.).

Aspects of the presently disclosed subject matter relate to a nucleic acid molecule encoding the antibody, antibody fragment or derivative thereof. As used interchangeably herein, the terms "nucleic acids," "oligonucleotides," and

"polynucleotides" include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term "nucleotide" as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term

"nucleotide" is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. The term "nucleotide" is also used herein to encompass "modified nucleotides" which comprise at least one of the following modifications: (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar. For examples of analogous linking groups, purine, pyrimi dines, and sugars, see for example PCT Patent App. Pub. No. WO 95/04064. The polynucleotide sequences of the presently disclosed subject matter may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. The term "polypeptide" or "protein" as used herein refers to a molecule comprising a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "polypeptide" may be used interchangeably. Proteins may be recombinant or naturally derived.

Following long-standing patent law convention, the terms "a," "an," and "the" refer to "one or more" when used in this application, including the claims. Thus, for example, reference to "a subject" includes a plurality of subjects, unless the context clearly is to the contrary (e.g., a plurality of subjects), and so forth. Throughout this specification and the claims, the terms "comprise,"

"comprises," and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. Likewise, the term "include" and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, parameters, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the presently disclosed subject matter. For example, the term "about," when referring to a value can be meant to encompass variations of, in some embodiments, ± 100% in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ±1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term "about" when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1 , 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration, and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

EXAMPLE 1

Materials and Methods

Strains and reagents

Strains and their properties are provided in Table 3. E. coli NEB 5 -alpha was used to create the library. Strain SNOBLA (SNO301 harboring pDIMC8-BLA) was used to perform band-pass selection. The E. coli K-12 strain MG1655 Alacl was used in all characterization experiments, except for those involving chromosomal reporters. All chemicals and culture media used were from Fisher Scientific or Sigma Aldrich. Enzymes were acquired from New England Biolabs. Oligonucleotides were purchased from Integrated DNA Technologies.

Plasmids construction

Plasmid descriptions are provided in Tables 4 and 5. The GBBP gene (proX; GenelD: 947165) without its signal peptide and stop codon was PCR-amplified from E. coli K12 genomic DNA such that HindlU and BamHl restriction enzyme sites were added at the 5' and 3' ends, respectively. The 945-bp PCR product was digested with HindlU and BamHl, and cloned into plasmid pUC19. The construct pUC/GBBP was the template to amplify a second copy of proX using primers that included in the 5' - end a restriction site for BamHl and two codons encoding two glycines. This second fragment was cloned into the pUC/GBBP BamHl digested plasmid, generating the construct pUC/GBBPlkGBBP containing a gene encoding an end-to-end fusion of the GBBP spanned by a linker GSGG The P102 constitutive promoter and TALELacOl was PCR-amplified from plasmid pBT102-TALE (Politz, et al, 2013) such that Notl md Kpnl restriction enzyme sites were added at the 5' and 3' ends, respectively. The 2627-bp PCR product was digested with Notl and Kpnl, and cloned into plasmid pTSl (Sohka, et al, 2009) replacing the lad gene. pLR plasmid was constructed by deleting lacl, gfp and tetR genes from pTSl using inverse PCR. The double mutated GB binding site (W140A, W188A) was constructed by inverse PCR. The BLA and sfGFP reporter plasmids were all built using plasmid pDIMC8 as a backbone via Gibson assembly. Constitutive promoters PI 00 and P106 were from the BioBrick J2310x series promoters (x: 0 or 6). The plasmids pCherryAlac, pl02TlysA, pBT102- TALE were previously described (Copeland, et al, 2016; Politz, et al, 2013).

Library creation by domain insertion

A custom MATLAB script was used to design all primer pairs and optimize the melting temperatures (Tra) to be close to 60^° C. Swiss-PdbViewer (Guex, et al, 1997) and Pymol (DeLano, et al, 2005) software were used to examine high- resolution crystal structure of GBBP (Schiefner, et al, 2004) (PDB identifier 1R9L) for residues that are solvent accessible, flexible, loosely packed, and between secondary structure elements to identify target sites to perform the circular permutation of GBBP. 137 positions were identified, and the circularly permuted genes were generated by PCR (Ribeiro, et al; Ribeiro, et al, 2015) using as a template pUC/GBBPlkGBBP. The desired 939-bp fragment was gel purified a 0.8% agarose gel in TAE buffer. Similar to previous studies (Ribeiro, et al, 2015; Tullman, et al, 2016) multiplex inverse PCR (Ribeiro, et al.) was used to linearize the construct pTSl-TALE in 194 specific codons of TALELacOl. The desired 8,103- bp fragment was gel purified. The linearized plasmid pTS 1-TALE was ligated to the cpGBBP amplicons.

The product of the ligation reaction was purified, concentrated and used to transform electrocompetent E. coli NEB 5 -alpha (New England Biolabs, Ipswitch, MA). After recovery, the cells were plated on LB-agar containing 50 Ng/mL spectinomycin (Sp) on bioassay plates (24.5 x 24.5 cm). After growth, all the bacterial colonies present on the plates were harvested in storage media (SOC + 10% glycerol (v/v)) and stored at -80^° C.

Selection of GB-activated TALE from a combinatorial library

Plasmid DNA was extracted from an aliquot of library cells and used to transform electrocompetent E. coli SNOBLA cells. In the negative selection, cells were plated on TB-agar (10 g tryptone, 5 g NaCl and 15 g agar per liter) plates containing 50 Ng/mL Sp, 50 Ng/mL streptomycin (Sm), 50 Ng/mL chloramphenicol (Cm), 20 Ng/mL tetracycline (Tet), 16 Ng/mL ampicillin (Amp) and 300 mM IPTG. Plates were incubated 20 h at 37 °C. Colonies that formed were recovered en masse in LB. In the positive selection, cells surviving the negative selection were spread on TB-agar plates containing Sp (50 Ng/mL), Sm (50 Ng/mL), Cm (50 Ng/mL), Amp (200 Ng/mL), 300 mM IPTG and 5 mM GB. Plates were incubated 20 h at 37 °C. Cultures inoculated with colonies that formed in the presence of GB were prepared and stored at -80° C. The sequences of the selected chimeras were obtained by Sanger sequencing.

MIC assay for Amp

Colonies that survived the two selection steps were assayed individually to analyze the MIC shift for Amp in the presence and absence of GB. A sample of frozen stocks was diluted to 3x105 CFU/mL and 30 NL was spread on TB-agar plates containing Sp (50 Ng/mL), Cm (50 Ng/mL) as well as 13 different Amp

concentrations (2-fold increments ranging from 0.25 mg/ml to 1,024 mg/ml). The plates were incubated at 37 °C for 20 h and further incubated at room temperature (23 °C) for 16 h to produce clearly visible growth. All samples were assayed in triplicate and the MIC was determined as the lowest Amp concentration with no visible growth on the plate.

Fluorescence measurements

Fluorescence measurements were used to estimate the protein production rates (P) as described previously (Copeland, et al, 2016; Leveau, et al, 2001; Ribeiro, et al, 2013). To evaluate the effect of GB on fluorescent protein expression,

Escherichia coli strain MG1655 Alacl harboring the appropriate expression and reporter plasmids was grown overnight at 37 °C on TB agar plates containing Sp and Cm. Five colonies were picked and used to inoculate TB liquid medium with antibiotics. The cultures were incubated 15 h at 30 °C / 250 r.p.m. The cultures were diluted 350-fold (ODeoo ~ 0.005) in fresh M63 minimal medium (15 mM (NH^SCM 22 mM KH2PO4 40 mM K2HPO4 25 μΜ FeS0₄, 2 mM MgS0₄, 0.1 mM CaCh, 5 mM Thiamine HC1, 0.2% (w/v) tryptone) containing 0.4% (w/v) glucose as the primary carbon source, Sp (50 Ng/mL) and Cm (50 Ng/mL). Cultures were induced with 10 mM GB (except where noted otherwise) and incubated for 2.5 h at 37 °C / 250 r.p.m. Cells harboring Lacl expression plasmids were induced with 500 NM IPTG instead of GB. A total of 150 NL of culture was transferred to two separate 96- well microplates, a 96-well clear bottom black plate for fluorescence and another 96- well plate for cell growth. The plates were incubated in microplate readers

(SpectraMax gemini xps and 384 plus, Molecular Devices, Sunnyvale, CA, USA) at 35 ^°C for 15 hr, during which the fluorescence and ODeoonm were measured every 20 min with constant agitation between readings. For superfolder GFP (sfGFP) and enhanced GFP (eGFP), excitation and emission detection were performed at 485/510 nm and 488/507 nm, respectively. For mCherry reporter protein excitation/emission wavelengths of 587/610 nm were used. Cells transformed with the same plasmids without the fluorescent protein were used as the negative control to correct for auto- fluorescence. Protein production rates (P) were calculated as described previously (Copeland, et al, 2016; Leveau, et al, 2001), and can be expressed by the following Equation 1 :

Where fss is derived from slope of the linear range of a plot of the fluorescence values as a function of ODeoonm, N is the cell growth rate, and m is the maturation constant for which values of 7.39 h^"1 and 0.739 h^"1 (Iizuka, et al, 2011 ; Pedelacq, et al., 2006) were used for sfGFP and eGFP, respectively. The slopes of the replicates were used to calculate the means and standard errors. P is given as relative fluorescence units per absorbance unit per hour

Lysine auxotrophy assay

E. coli K-12 strain MG1655 harboring the expression vectors were grown overnight at 37 °C on TB agar plates in TB-agar with 50 Ng/mL kanamycin (kan). Five colonies were picked and used to inoculate TB liquid medium containing kan (50 Ng/mL) and 0.4 mM lysine. Cultures were incubated 15 h at 30 °C / 250 r.p.m. Saturated cultures were centrifugation (5 min at 4,000g), and the cell pellet was resuspended in an equivalent volume of M63 minimal medium (without tryptone) containing 0.4% (w/v) glucose as the primary carbon source and antibiotic. Cultures were then diluted to an ODeoonm 0.005 in same M63 minimal medium. Cultures were incubated for 2.5 h at 37 °C / 250 r.p.m. and then induced with 5 mM GB (except where noted otherwise). A total of 150 NL of culture was transferred to a 96-well microplate. The plates were incubated in a microplate reader (SpectraMax, Molecular Devices, Sunnyvale, CA, USA) at 37 ^°C for 18 h, during which the ODeoonm was measured every 20 min with constant agitation between readings. All samples were assayed in quintuplicate and the mean of the five values was used for subsequent comparisons. EXAMPLE 2

RESULTS

Strategy for the creation of a Betaine-Inducible Transcriptional Effector

An inducible repressor was created by fusion of a TALE and a ligand binding protein that binds a desired inducer. Allosteric protein switches can be created by fusing two domains in such a way that the activity of the output domain is regulated by the input domain's recognition of an input signal (Ostermeier, 2005; Stein, et al, 2015). Domain insertion has been shown to be an effect method of establishing this coupling of activities. Domain insertion combined with circular permutation of the insert domain has been used to create switches with very large differences in activity between their "on" and "off states (Guntas, et al, 2005), but a suitable selection or screen is necessary to identify those rare fusions that behave as switches. TALElacOl was selected as the input domain - a TALE that was designed to bind the lacOl operator and repress expression from the trc promoter in E. coli (Politz, et al, 2013). First, a combinatorial library was created of fusions of the genes encoding

TALElacOl and glycine betaine binding protein (GBBP), a periplasmic binding protein that undergoes a large conformation change upon binding GB (Schiefner, et al, 2004). GB (i.e., 2-trimethylammonioacetate) is a low cost osmolyte that crosses the plasma membrane, can accumulate at high levels intracellularly, and is not metabolized by E. coli. Subsequently, the library was subjected to selective pressure for the identification of chimeric proteins in which the TALElacOl domain's ability to repress expression could be alleviated by the presence of GB. This process resulted in a family of Betaine-Inducible Transcriptional Effectors (BITE) (FIG. 3 A) that repress gene expression in the absence of GB but not in the presence of GB.

Library creation by insertion of circularly permuted GBBP (cpGBBP) into

TALElacOl

The TALElacOl gene was previously codon-optimized for expression in E. coli (Politz, et al, 2013). This protein was designed to bind 18 base pairs of the lacOl operator. The ability of TALElacOl to repress the expression of the reporter protein TEM-1 β-lactamase (BLA) when placed under control of the tac promoter on plasmid pDIM-C8-BLA was tested. The tac promoter is a hybrid of the trp and lac promoters and contains the lacOl operator (Deboer, et al, 1983). The TALElacOl gene was placed in the vector pTS l (Sohka, et al., 2009) under the control of the strong constitutive promoter PI 02 to create pTSl-TALE. As expected, cells harboring pDIM-C8-BLA and pTSl-TALE could not grow in the presence of Amp (FIG. 9 A) consistent with TALElacOl binding to the lacOl operator and preventing transcription initiation.

A library of GBBP-TALElacOl fusions (FIG. 10) to be subjected to a two- tiered selection for inducible-repressor activity was constructed. A library encoding 137 different circularly permuted GBBP proteins (cpGBBP) in which the original N- and C-termini were joined by a GSGG peptide linker designed to be of sufficient length to connect the termini without perturbing GBBP structure also was constructed. These circular permutation sites were at locations that are solvent accessible, flexible, loosely packed, and between secondary structure elements. The cpGBBP DNA also encoded two random amino acids at each new termini to allow for some space between the two protein domains and to alleviate possible disturbances caused by insertion. The cpGBBP DNA was inserted at 194 positions in TALElacOl in pTSTALE (FIGS. 10 and 11). Sites were chosen that were not expected to completely disrupt the TALE domain's ability to bind DNA. The naive library was comprised of 2.8 ^χ 10⁵ transformants, of which approximately 60% contained the cpGBBP inserted at sites that were well distributed throughout TALElacOl.

Selection of inducible repressors

A band pass filter gene circuit (Sohka, et al, 2009) facilitated identification of inducible repressors from the library. This circuit provides the ability to select cells that have a certain level of ampicillin resistance between an upper and a lower threshold (FIGS. 3B, 3C, and 3D). In this selection system, a sublethal level of the ampicillin is required to induce the expression of tetracycline resistance gene. In the presence of tetracycline and low ampicillin concentration, cell growth requires a low level of β-lactamase expression. This level is high enough to maintain the ampicillin below lethal levels, but not too high to eliminate the signal necessary for induction of tetracycline resistance. When β-lactamase expression is repressed by TALElacOl and Tet is present, cells grow only at low Amp concentrations. Plating in the presence of Tet and low Amp concentrations is described herein as a "negative" selection in the sense that it selects for the absence of high Amp resistance. Selection for growth at high Amp is a positive selection. Thus, the band-pass circuit could be used to select TALElacOl-cpGBBP fusions that repress BLA expression in the absence of GB (negative selection) and lose the ability to repress in the presence of GB (positive selection). A total of ~1 ^χ 10⁸ colonies were plated under negative selection conditions (16 Ng/mL Amp and 20 Ng/mL Tet) in the absence of GB and obtained on the order of 1000 colonies. Next, cells obtained from these colonies were plated under positive selection conditions (200 Ng/mL Amp) in the presence of 5 mM GB. Clones from the positive selection step were screened individually for a higher MICAmp in the presence of GB compared to its absence. Sequencing of hits resulted in the identification of seven unique sequences encoding full-length fusions of TALELacOl and GBBP (FIG. 11 and Table 2). The seven plasmids were transformed into fresh cells and found that GB caused a twofold to eightfold increase in MICAmp (Table 2). The fusion BITElacO-C7 conferred the largest inducible effect (eightfold) in the presence of GB.

In this fusion, circular permutation split the GBBP domain between residues

130 and 131 (numbered according to the original GBBP protein), which are located in a loop between a β-strand and a a-helix. cpGBBP was inserted in the conserved N- terminal region of the TALE backbone in TALElacOl, which was a common insertion region in the selected fusions (FIGS. 11 and 12). In liquid media, cells expressing BITElacO-C7 required 10 mM GB for full induction of Amp resistance (FIGS. 13A), so this concentration of GB was used for all subsequent experiments unless otherwise indicated.

Inducible control of expression from plasmids and the chromosome

BITElacO-C7's ability to act as a GB-inducible repressor in plasmidial and chromosomal contexts was evaluated using MG1655 Alacl E coli cells harboring a high copy number plasmid for constitutively expressing BITElacO-C7 (FIG. 4A). To compare BITElacO-C7/GB with the canonical LacI/IPTG system, BITElacO-C7 was replaced with lad. Three plasmidial reporters were tested: β-lactamase (BLA), superfolder GFP (sfGFP) (Pedelacq, et al, 2006) and mCherry. All three genes were placed under the control of the strong inducible tac promoter containing the lacOl operator 3' of the -10 sequence to which TALElacOl binds. A low copy plasmid served as the host for BLA and sfGFP and a medium copy plasmid was the host for mCherry. For the chromosomal reporter system, a MG1655 Alacl derived strain with the sfGFP gene placed under the control of the native lac promoter was used. For the BLA reporter, expression was quantified by measuring the MICAmp, and for fluorescent protein expression the rate of production of fluorescence was measured as described (Leveau, et al., 2001).

As desired, TALElacOl and BITElacO-C7 repressed expression in all systems, but GB increased expression only in combination with BITElacO-C7 (FIGS. 4B, 4C, and 4D and FIG. 14A). BITElacO-C7 repressed expression as effectively as TALElacOl for the lac promoter on the chromosome (FIG. 4D) and for the tac promoter on a medium-copy plasmid (FIG. 14 A), but not as effectively for the tac promoter on a low-copy plasmid (FIG. 4B and 4C). BITElacO-C7's decreased ability to repress in some scenarios suggests that insertion of cpGBBP decreased the TALElacOl domain's affinity for DNA. In the presence of GB, BITElacO-C7- achieved the same or nearly the same level of expression as that of the fully induced LacI-IPTG system (FIG. 4B, 4C, and 4D and FIG. 14A). GB increased expression from 9.8 ± 4.0 fold to 148 ± 55 fold depending on the promoter and its location. A similar dynamic range was seen for the LacI-IPTG system (4.0 ± 0.4 to 154 ± 45 fold), but in different context. BITElacO-C7's fold difference in expression upon induction was inferior to that of Lacl for tac on the low-copy plasmid, equivalent for tac on the medium-copy plasmid, and superior for lac on the chromosome. Lad's relatively poor performance on the chromosome stemmed from its poor repression, as previously observed when Lacl was expressed from the same constitutive promoter used here (PI 02). Copeland et al. (2016).

The cpGBBP domain of BITELacO-C7 was then mutated to provide evidence that BITElacO-C7 binding to GB is responsible for the loss of repression. The W188A mutation (numbering as in the GBBP protein) was introduced, which weakens, but does not eliminate GB binding (Schiefher, et al, 2004). As expected, a higher extracellular concentration of GB (>10x) was required for full alleviation of the repression by BITElacOC7mut (FIG. 13B). A similar effect was observed when β-lactamase was used as output (Table S2).

Altogether, these results support the argument that the BITELacO-C7 functions as an inducible repressor in which GB binding to its cpGBBP domain decreases the TALELacOl domain's affinity for DNA, thereby alleviating repression of plasmidial and chromosomal promoters that possess the lacOl operator.

Conversion of constitutive promoters into inducible promoters

Further, it was contemplated whether expression from constitutive promoters could be controlled by BITElacO-C7 if the lacOl operator site was introduced. Single or multiple lacOl TALE binding sites (TBS) were introduced between the reporter genes (sfGFP or BLA) and the PI 00 and PI 06 constitutive promoters, which have the following relative strengths: 1.0 and 0.47. The introduction of a single TBS changed the promoters into ones inducible by GB, but to different degrees depending on the promoter's strength (FIG. 5A).

BITELacO-C7 could better repress the expression of sfGFP from the weaker promoter (P106), resulting in a 17.9 ± 2.1 fold increase in expression in the presence of GB (FIG. 5A). Similar GB dependent changes in expression were observed when sfGFP was replace with the gene encoding enhanced GFP (eGFP) (Tsien, 1998) (the fold changes were 6.3 ± 2.4 with P 100 and 23.0 ± 16.5 with P 106) (FIG. 14B). With BLA as the reporter, the BITELacO-C7/GB combination also showed a larger dynamic range with the weaker promoter due to better repression (FIG. 5B). These results suggest that larger dynamic ranges are best achieved by combining

BITELacO-C7 with weaker promoters.

Increase in the dynamic range through multiple TBS

Without wishing to be bound to any one particular theory, it was thought that another way to increase the dynamic range would be to increase repression through the introduction of multiple TBS. The effect of 1, 2, 3 and 4 TBS between the P106 constitutive promoter and the bla reporter gene was compared (FIG. 5B, FIG. 15, Table 7). Although increasing the number of TBS slightly decreased expression in the presence of GB, it decreased expression in the absence of GB more, increasing the dynamic range from 32-fold (1 TBS) to 128-fold (4 TBS). The range observed with BITELacO-C7 and four TBS (128-fold) was close to the dynamic range of the canonical repressors system LacI-IPTG (256-fold; FIG. 4B). As expected, four TBS required higher levels of GB for induction (FIG. 13C) with apparent i^d's of 0.85 mM for one TBS and 3.2 mM for 4 TBS. These findings indicate that BITElacO-C7's performance can be optimized by the introduction of multiple TBS sites to increase the dynamic range of expression.

Modularity of BITE inducible repressors

TALE proteins were selected as the DNA binding motif for the presently disclosed engineered inducible repressors due to their DNA binding modularity. Again, without wishing to be bound to any one particular theory, it was thought that BITELacO-C7 would share this modularity, thus allowing researchers to inducibly repress endogenous E. coli promoters simply through the appropriate modifications of the TALE domain to make it bind the target promoter at a site that would repress expression. To test the modularity of the presently disclosed system, the TALE domain of BITELacO-C7 was replaced with one targeting 22 base pairs in the lysA promoter (Plys) to create BITElysA (FIG. 7A). The Plys promoter controls the expression of LysA that encodes a diaminopimelate decarboxylase that is essential for growth on minimal medium lacking lysine (Dewey, et al, 1952). Copeland et al, 2016 showed that the TALELysA protein targeting these 22 bp represses Plys and prevents growth in minimal media lacking lysine. Cell outgrowth was monitored on minimal medium to test the auxotrophic recovery in the presence of GB. Cells expressing TALElysA did not grow for nearly 20 h after inoculation regardless of the presence of GB, confirming that TALElysA is an effective repressor of Plys (FIG. 7B). Copeland et al. (2016). Growth could be rescued by the addition of lysine to the media. In the absence of GB, cells expressing BITElysA exhibited slow growth that plateaued at low cells densities. The addition of GB restored the growth kinetics to that observed with supplementation of lysine to the media. The addition of GB to the growth media of cells expressing BITElysA caused a shorter lag phase (7.8 h vs. 9.5 hours), a 37% higher initial growth rate (0.84 ± 0.28 h-1 vs. 1.08 ± 0.13 h-1), a 4.8- fold higher growth rate between 15-17 hours (0.15 ± 0.01 h-1 vs. 0.04 ± 0.02 h-1), and a 6-fold higher cell density after 18 h of culturing. These results indicate that BITElysA is an inducible repressor of Plys, but one that cannot fully repress LysA expression in the absence of GB. It is thought that low-levels of the enzyme LysA is enough to provide some lysine to the cell, just not enough for robust growth. These data demonstrate that BITE inducible repressors have the potential to be reprogramed to inducibly control expression from arbitrarily -chosen promoters in E. coli. EXAMPLE 3

DISCUSSION

Reprogrammable frara-acting regulatory tools are powerful components for building orthogonal and multiplexed regulatory systems. BITELacO-C7 is such a tool. Its repression of promoters can be by modifications to the TALE domain or modifications to the promoter. The combination of BITELacO-C7 and GB can control the expression of genes from inducible and constitutive promoters on plasmids and on the chromosome.

It is contemplated that BITE proteins can be adapted to work in other bacterial or eukaryote hosts, as has been done with other canonical ligand-dependent repressor systems from bacteria, such as TetR, which was adapted for mammalian cells

(Gossen, et al, 1992). Escherichia coli and mammalian cells can synthesize GB in the presence of exogenous choline (Lamark, et al, 1991; Wilken, et al., 1970).

Therefore, the cellular synthesis of GB can be avoided using a chemically defined minimal medium. GB is an osmoprotectant molecule and is synthetized specially in hyperosmotic condition. In mammalian cells, such as liver and kidney cells the rate of its synthesis is not affected by hyperosmolarity. Rather, hyperosmolarity increases the number of GB transporters (Burg, et al, 2008).

BITELacO-C7 caused a greater fold induction with GB when regulated genes were integrated on the chromosome or on a low copy number plasmid under a weak promoter with multiple TALE binding sites. This result is in accord with previous studies. A study involving a riboswitch and TALE construction demonstrated that when the target gene was under control of a weaker promoter a higher modulation was achieved (Rai, et al., 2015). In a study in which TALE repression was alleviated by proteolysis, a higher fold induction for a chromosomal reporter was achieved by the variants that showed a greater repression (Copeland, et al, 2016).

The non-canonical repeats at the N-terminus of TALEs play a key role in TALE binding to DNA (Meckler, et al, 2013; Rogers, et al, 2015; Gao, et al., 2012; Lamb, et al, 2013) and were the site of insertion of the cpGBBP in BITELacO-C7. In TALE proteins, this region nonspecifically interacts with DNA and serves as an essential "nucleation site" without which TALE binds poorly to DNA (Gao, et al,

2012) . In a previous study, the introduction of a protease cleavage site into the N- terminus of TALElacOl allowed a protease to regulated its repression of gene expression. Copeland et al. (2016). These studies and the presently disclosed subject matter suggest the N-terminus of TALE is hotspot region to generate allosteric coupling and modulate DNA binding without disrupting TALE specificity.

At least two general mechanisms could be invoked to explain BITELacO-C7's function. TALE proteins adopt an extended conformation during the DNA target search process and a compressed conformation when bound to the DNA target (Cuculis, et al, 2015; Wan, et al, 2013). GBBP adopts at least two different conformations: a ligand-free open form, and a closed ligand-bound form. Without wishing to be bound to any one particular theory, it is thought that the GB-sensitive conformational change in GBBP coupled to the conformational plasticity of TALE may contribute to the transduction of the GB input signal to modulate the TALE domain's DNA affinity in a manner analogous to heterotropic allosteric proteins. An alternative mechanism in which GB regulates the cellular accumulation of

BITELacO-C7, as seen in some switches built by domain insertion (Choi, et al,

2013) , seems less likely. Ligand binding is a stabilizing interaction that would be expected to cause increased accumulation, not decreased accumulation as would be required for induction with GB. However, GB binding could cause a conformational change that makes the protein less proteolytically stable, thereby decreasing its cellular accumulation and repression. Although such a mechanism is not canonical allostery, it can be thought of as a kind of allostery in which ligand binding modulates the proteases accessibility at some distal site. Future characterization of the BITE proteins will provide a better mechanistic understanding and inform studies to expand their usefulness.

EXAMPLE 4

REPRESENTATIVE SEQUENCES

TALE binding regions;

lac operator - AATTGTGAGC GGAT AAC AATT (SEQ ID NO: 1)

lysA operator - TCTTTTTATGATGTGGCGTAATC (SEQ ID NO: 2)

Protein sequence of the BITEs variants

M: start codon (methionine)

Blue: conserved N-terminal regions of the TALE backbone

Black: variable regions of the TALE backbone

Orange: GBBP domain

Purple: linker

Red: conserved C-terminal regions of the TALE backbone

Bold black: FLAG-tag BITElacO-04

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVVAIASN GGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA SNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQALETVQR LLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLADLPGKGITVNP VQSTITEETFQTLLVSRALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAV NWTPLHDNMYEAAGGDKKFYREGVFVNGAAQGYLIDKKTADQYKITNIA QLKDPKIAKLFDTNGDGKADLTGCNPGWGCEGAINHQLAAYELTNTVTH NQGNYAAMMADTISRYKEGKPVFYYTWTPYWVSNELKPGKDVVWLQVP FSALPGDKNADTKLPNGANYGFPVSTMHIVANKAWAEKNPAAAKLFAIM QLPVADINAQNAIMHDGKASEGDIQGHVDGWIKAHQQQFDGWVNEALA AQKCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVA IASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVV AIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQV VAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPALESIV AQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRR IPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSDYKDDDD

K (SEQ ID NO: 3)

BITElacO-14

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLNLTPEQVV AIASN GGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPV LCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIA SNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVA IASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNHGGKQALETVQR LLPVLCQAPSEVDYNVGYTSLASGDATFTAVNWTPLHDNMYEAAGGDKK FYREGVFVNGAAQGYLIDKKTADQYKITNIAQLKDPKIAKLFDTNGDGKA DLTGCNPGWGCEGAINHQLAAYELTNTVTHNQGNYAAMMADTISRYKEG KPVFYYTWTPYWVSNELKPGKDVVWLQVPFSALPGDKNADTKLPNGAN YGFPVSTMHIVANKAWAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKA SEGDIQGHVDGWIKAHQQQFDGWVNEALAAQKGSGGADLPGKGITVNPV QSTITEETFQTLLVSRALEKLGYTVNKYHGLTPEQVVAIASNNGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTP EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPA LESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKR TNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSDYK DDDDK (SEQ ID NO: 4)

BITElacO-22

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAGTEGVFVNGAAQGY LIDKKTADQYKITNIAQLKDPKIAKLFDTNGDGKADLTGCNPGWGCEGAI NHQLAAYELTNTVTHNQGNYAAMMADTISRYKEGKPVFYYTWTPYWVS NELKPGKDVVWLQVPFS ALPGDKNADTKLPNGANYGFPVSTMHIVANKA WAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKASEGDIQGHVDGWIKA HQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQSTITEETFQTLLVSR ALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNWTPLHDNMYEAAGG DKKFYRRHPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VV AI ASNNGGKQ ALETVQRLLPVLCQ AHGLTPEQVV AI ASNGGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP EQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQ ALETVQRLLPVLCQ AHGLTPEQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNG GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQF GMSDYKDDDDK (SEQ ID NO: 5) BITElacO-H7

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAGTEGVFVNGAAQGY LIDKKTADQYKITNIAQLKDPKIAKLFDTNGDGKADLTGCNPGWGCEGAI NHQLAAYELTNTVTHNQGNYAAMMADTISRYKEGKPVFYYTWTPYWVS NELKPGKDVVWLQVPFSALPGDKNADTKLPNGANYGFPVSTMHIVANKA WAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKASEGDIQGHVDGWIKA HQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQSTITEETFQTLLVSR ALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNWTPLHDNMYEAAGG DKKFYRLNPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTP EQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPEQVV AIASHDGGKQ ALETVQRLLPVLCQ AHGLTPEQVV AIASNIGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVV AIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNG GGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHA PALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFG MSDYKDDDDK (SEQ ID NO: 6)

BITElacO-C7

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG PPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLVQKADLTGCNPG WGCEGAINHQLAAYELTNTVTHNQGNYAAMMADTISRYKEGKPVFYYT WTPYWVSNELKPGKDVVWLQVPFSALPGDKNADTKLPNGANYGFPVST MHIVANKAWAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKASEGDIQG HVDGWIKAHQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQSTITEE TFQTLLVSRALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNWTPLHD NMYEAAGGDKKFYREGVFVNGAAQGYLIDKKTADQYKITNIAQLKDPKI AKLFDTNGDGIHNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTP EQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAH GLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGG KQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQ AHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIG GKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLC QAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI GGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVL CQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NGGGRP ALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLP HAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQ FGMSDYKDDDDK (SEQ ID NO: 7)

BITElacO-G10

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGPVST MHIVANKAWAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKASEGDIQG HVDGWIKAHQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQSTITEE TFQTLLVSRALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNWTPLHD NMYEAAGGDKKFYREGVFVNGAAQGYLIDKKTADQYKITNIAQLKDPKI AKLFDTNGDGKADLTGCNPGWGCEGAINHQLAAYELTNTVTHNQGNYA AMMADTISRYKEGKPVFYYTWTPYWVSNELKPGKDVVWLQVPFSALPGD KNADTKLPNGANYGFHRELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAW RNALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQV VAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETV QRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQA LETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGL TPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAH GLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGK QALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGG GRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAP ALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGM SDYKDDDDK (SEQ ID NO: 8)

BITElacO-D2

MVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPA ALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRG TGCNPGWGCEGAINHQLAAYELTNTVTHNQGNYAAMMADTISRYKEGKP VF YYTWTP YWV SNELKP GKD V VWLQ VPF S ALP GDKN ADTKLPNGANYG FPVSTMHIVANKAWAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKASE GDIQGHVDGWIKAHQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQS TITEETFQTLLVSRALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNWT PLHDNMYEAAGGDKKFYREGVFVNGAAQGYLIDKKTADQYKITNI AQLK DPKIAKLFDTNGDGKADLRPPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN ALTGAPLNLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVV AIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQ RLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQ VVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALET VQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPE QVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALE TVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTP EQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQAL ETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLT PEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGGGRPA LESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKR TNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDDAMTQFGMSDYK DDDDK (SEQ ID NO: 9) BITElvsA

MHHHHHHVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIV ALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLT VAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLVQKAD LTGCNPGWGCEGAINHQLAAYELTNTVTHNQGNYAAMMADTISRYKEGK PVFYYTWTPYWVSNELKPGKDVVWLQVPFSALPGDKNADTKLPNGANY GFPVSTMHIVANKAWAEKNPAAAKLFAIMQLPVADINAQNAIMHDGKAS EGDIQGHVDGWIKAHQQQFDGWVNEALAAQKGSGGADLPGKGITVNPVQ STITEETFQTLLVSRALEKLGYTVNKPSEVDYNVGYTSLASGDATFTAVNW TPLHDNMYEAAGGDKKFYREGVFVNGAAQGYLIDKKTADQYKITNI AQL KDPKIAKLFDTNGDGIHNLTPEQVVAIASNGGGKQALETVQRLLPVLCQA HGLTPEQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNGG GKQALETVQRLLPVLCQAHGLTPEQVVAIASNNGGKQALETVQRLLPVLC QAHGLTPEQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNN GGKQ ALETVQRLLP VLCQ AHGLTPEQVV AIASHDGGKQ ALETVQRLLPVL CQAHGLTPEQVVAIASNHGGKQALETVQRLLPVLCQAHGLTPEQVVAIAS NNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNI GGKQ ALETVQRLLP VLCQAHGLTPEQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQRLL PVLCQAHGLTPEQVV AIASHDGGKQ ALETVQRLLPVLCQAHGLTPEQVV A IASNI GGKQ ALETVQRLLPVLCQAHGLTPEQVVAIASNI GGKQ ALETVQRL LPVLCQAHGLTPEQVVAIASNGGGKQ ALETVQRLLPVLCQAHGLTPEQVV AIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVK KGLPHAPALIKRTNRRIPERTSHRVADHAQVVRVLGFFQCHSHPAQAFDD AMTQFGMSDYKDDDDK (SEQ ID NO: 10)

REFERENCES

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Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. A modular, inducible repressor for control of gene expression, the repressor comprising a DNA binding protein comprising a transcription activator-like effector (TALE) fused to a protein that binds to an exogenous inducer molecule comprising glycine betaine binding protein (GBBP); wherein the repressor is directly inducible by the exogenous inducer molecule.

2. The modular, inducible repressor of claim 1, wherein the exogenous inducer molecule comprises glycine betaine.

3. The modular, inducible repressor of claim 1, wherein the repressor comprises a betaine-inducible transcriptional factor (BITE).

4. The modular, inducible repressor of claim 3, wherein the betaine- inducible transcriptional factor (BITE) is selected from a protein having a sequence at least 90% identical to any one of BITElacO-04 (SEQ ID NO: 3), BITElacO-14 (SEQ ID NO: 4), BITElacO-22 (SEQ ID NO: 5), BITElacO-H7 (SEQ ID NO: 6),

BITElacO-C7 (SEQ ID NO: 7), BITElacO-GlO (SEQ ID NO: 8), BITElacO-D2 (SEQ ID NO: 9), and BITElysA (SEQ ID NO: 10).

5. The modular, inducible repressor of claim 3, wherein the betaine- inducible transcriptional factor (BITE) is selected from a protein having a sequence at least 95% identical to any one of BITElacO-04 (SEQ ID NO: 3), BITElacO-14 (SEQ ID NO: 4), BITElacO-22 (SEQ ID NO: 5), BITElacO-H7 (SEQ ID NO: 6),

BITElacO-C7 (SEQ ID NO: 7), BITElacO-GlO (SEQ ID NO: 8), BITElacO-D2 (SEQ ID NO: 9), and BITElysA (SEQ ID NO: 10).

6. The modular, inducible repressor of claim 3, wherein the betaine- inducible transcriptional factor (BITE) is selected from a protein having a sequence at least 100% identical to any one of BITElacO-04 (SEQ ID NO: 3), BITElacO-14 (SEQ ID NO: 4), BITElacO-22 (SEQ ID NO: 5), BITElacO-H7 (SEQ ID NO: 6), BITElacO-C7 (SEQ ID NO: 7), BITElacO-GlO (SEQ ID NO: 8), BITElacO-D2 (SEQ ID NO: 9), and BITElysA (SEQ ID NO: 10).

7. A method of controlling gene expression in a cell, the method comprising contacting the cell with a modular, inducible repressor of any one of claims 1-6.

8. A biosensor comprising a modular, inducible repressor of any one of claims 1-6.