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WO2018129021A1 - Répresseurs modulaires, inductibles pour la régulation de l'expression génique - Google Patents

Répresseurs modulaires, inductibles pour la régulation de l'expression génique Download PDF

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WO2018129021A1
WO2018129021A1 PCT/US2018/012159 US2018012159W WO2018129021A1 WO 2018129021 A1 WO2018129021 A1 WO 2018129021A1 US 2018012159 W US2018012159 W US 2018012159W WO 2018129021 A1 WO2018129021 A1 WO 2018129021A1
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bitelaco
inducible
tale
seq
protein
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Marc Ostermeier
Lucas Ferreira RIBEIRO
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Johns Hopkins University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • 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 ⁇ -D-l-thiogalactopyranoside (IPTG) and anhydrotetracycline, respectively, which makes the use of these natural repressors economically unfeasible on an industrial scale.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • 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.
  • 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).
  • heterotropic allosteric proteins e.g., Lacl
  • 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).
  • a conditional repressor i.e., the inducer causes derepression
  • an inducible repressor i.e., the inducer causes derepression
  • 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;
  • the repressor is directly inducible by the exogenous inducer molecule.
  • 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).
  • the DNA binding protein comprises a transcription activator-like effector (TALE).
  • the protein that binds to an exogenous inducer molecule comprises glycine betaine binding protein (GBBP).
  • the exogenous inducer molecule comprises glycine betaine.
  • presently disclosed modular, inducible repressor comprises a betaine-inducible transcriptional factor (BITE).
  • BITE betaine-inducible transcriptional factor
  • 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,
  • 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.
  • the presently disclosed subject matter provides a biosensor comprising a presently disclosed modular, inducible repressor.
  • 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.
  • FIGS. 2 A and 2B show (A) scheme of the heterodimerization.
  • 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:
  • 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).
  • TBS TALE binding site located between the promoter and the ribosome binding site (RBS).
  • BITE binding represses transcription of the target gene.
  • BITE glycine betaine
  • 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 ⁇ -lactamase (BLA) activity for hydrolysis of ampicillin (Amp), cell wall synthesis is compromised and cells cannot proliferate.
  • BLA ⁇ -lactamase
  • 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
  • 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.
  • FIGS. 5 A and 5B show the characterization of BITElacO-C7 controlling constitutive promoters with single or multiple TBS.
  • 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).
  • TBS TALE binding sites
  • 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.
  • 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 10 4 cells) were spotted onto plates containing the presence or absence of 200 ⁇ g/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;
  • 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 ⁇ g/mL Amp in which BITElacO-C7 regulates ⁇ -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.
  • 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.
  • BITElacO-C7 regulates ⁇ -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 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 provides modular, inducible repressors for the control of gene expression.
  • 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.
  • the presently disclosed modular, inducible repressors can be used as tools to control gene expression (new vectors), biosensors, and gene therapy tools.
  • Transcription activator-like effectors 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.
  • 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.
  • 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.
  • GBBP Escherichia coli glycine betaine binding protein
  • BITE Betaine-Inducible Transcriptional Effector
  • 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.
  • 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
  • TALEs Transcriptional Activator-Like Effectors
  • 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).
  • RVD Repeat Variable Di-Residues
  • 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.
  • 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.
  • TALE is used to co-localize an inducible system to a desire promoter region.
  • FIG. 2A 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.
  • TALE is designed to repress gene expression and protease recognition sites are inserted into TALE backbone (FIG. 2B).
  • FPGA backbone TALE backbone
  • 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.
  • 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.
  • BITE Betaine-Inducible Transcriptional Effector
  • TALE non-homologous gene
  • GBBP GBBP
  • 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
  • cpGBBPs circularly permuted GBBPs
  • a TALElacO gene was synthesized and codon-optimized for expression in E. coli by the Vietnameser 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)).
  • Plasmid pDIMC8-BLA contains the TEM-1 ⁇ - 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a repressor protein binds to the operator region of a promoter and blocks RNA polymerase, thereby repressing gene expression.
  • TALElacO was designed by the Vietnameser 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.
  • ⁇ -lactamase bla
  • mCherry Two plasmidial reporters were tested: ⁇ -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 ⁇ -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
  • a fluorescent reporter gene (sfGFP) contain a LacOl sequence upstream was integrated into the lacIZYA locus and targeted it with BITElacO-C7.
  • sfGFP fluorescent reporter gene
  • FIG. 4C a weak sfGFP production was observed (FIG. 4C) consistent with repression of the gene.
  • the presence of GB resulted in a significant increase of production of the sfGFP consistent with a derepression and high-level expression of sfGFP.
  • the insertion of the GBBP domain in conserved N-ter of TALE would allow the presently disclosed BITE system to keep its modularity.
  • 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.
  • the BLA gene also showed higher modulation when the gene was controlled by weaker promoter (P 700 : 8x and V 106 : 16x) (FIG. 6B).
  • P 700 : 8x and V 106 : 16x weaker promoter
  • This effect can be explained by leaky expression when a stronger promoter was used, decreasing the difference between "on” and "off states.
  • the TBS number was increased downstream the P 106 constitutive promoter.
  • 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.
  • 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 are site-specific DNA-binding proteins that can be reprogramed to specifically interact with any desired DNA sequence target.
  • 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.
  • BITE Betaine-Inducible Transcriptional Effectors
  • 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.
  • the presently disclosed BITE system can be used as a biosensor.
  • 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.
  • plasmids could be developed with BITE controlled promoters as alternatives to commonly used inducible promoters. II. GENERAL DEFINITIONS
  • 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.
  • 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 AlignTM program of the
  • nucleic acid molecules encoding the antibody, antibody fragment or derivative thereof.
  • nucleic acids 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polypeptide or “protein” as used herein refers to a molecule comprising a string of at least three amino acids linked together by peptide bonds.
  • protein and “polypeptide” may be used interchangeably. Proteins may be recombinant or naturally derived.
  • 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.
  • E. coli NEB 5 -alpha was used to create the library.
  • Strain SNOBLA SNO301 harboring pDIMC8-BLA
  • 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.
  • 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).
  • 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.
  • 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.
  • Plasmid DNA was extracted from an aliquot of library cells and used to transform electrocompetent E. coli SNOBLA cells.
  • 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.
  • 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.
  • 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 ⁇ FeS0 4 , 2 mM MgS0 4 , 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.
  • 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
  • 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
  • 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.
  • 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).
  • a combinatorial library was created of fusions of the genes encoding
  • TALElacOl and glycine betaine binding protein a periplasmic binding protein that undergoes a large conformation change upon binding GB (Schiefner, et al, 2004).
  • GB i.e., 2-trimethylammonioacetate
  • GB is a low cost osmolyte that crosses the plasma membrane, can accumulate at high levels intracellularly, and is not metabolized by E. coli.
  • 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.
  • BITE Betaine-Inducible Transcriptional Effectors
  • 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 ⁇ -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 ⁇ 10 5 transformants, of which approximately 60% contained the cpGBBP inserted at sites that were well distributed throughout TALElacOl.
  • 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).
  • a sublethal level of the ampicillin is required to induce the expression of tetracycline resistance gene.
  • cell growth requires a low level of ⁇ -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.
  • a total of ⁇ 1 ⁇ 10 8 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.
  • 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.
  • 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).
  • TALElacOl a common insertion region in the selected fusions (FIGS. 11 and 12).
  • 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.
  • 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: ⁇ -lactamase (BLA), superfolder GFP (sfGFP) (Pedelacq, et al, 2006) and mCherry.
  • BLA ⁇ -lactamase
  • sfGFP superfolder GFP
  • mCherry 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.
  • a MG1655 Alacl derived strain with the sfGFP gene placed under the control of the native lac promoter was used.
  • 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).
  • 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.
  • 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).
  • TBS lacOl TALE binding sites
  • 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
  • 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).
  • 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.
  • 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
  • GB is an osmoprotectant molecule and is synthetized specially in hyperosmotic condition.
  • 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).
  • 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).
  • this region nonspecifically interacts with DNA and serves as an essential "nucleation site" without which TALE binds poorly to DNA (Gao, et al,
  • 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.
  • 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.
  • TALE transcription activator-like effector
  • TALE protein dynamics reveals a two-state search mechanism. Nat Commun,

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Abstract

L'invention concerne un répresseur modulaire, inductible pour la régulation de l'expression génique. De manière générale, le répresseur comprend une protéine de liaison à l'ADN fusionnée à une protéine qui se lie à une molécule inductrice exogène, où le répresseur est directement inductible par la molécule inductrice exogène. Dans des modes de réalisation particuliers, la protéine de liaison à l'ADN comprend un effecteur de type activateur de transcription (TALE) et la protéine qui se lie à une molécule inductrice exogène comprend une protéine de liaison à la glycine bétaïne (GBBP). Dans ces modes de réalisation, la molécule inductrice exogène comprend la glycine bétaïne.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011146121A1 (fr) * 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Nouvelles protéines se liant à l'adn et leurs utilisations
WO2015185691A9 (fr) * 2014-06-05 2016-03-31 Eth Zurich Système de biocapteur cellulaire à faible fuite

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011146121A1 (fr) * 2010-05-17 2011-11-24 Sangamo Biosciences, Inc. Nouvelles protéines se liant à l'adn et leurs utilisations
WO2015185691A9 (fr) * 2014-06-05 2016-03-31 Eth Zurich Système de biocapteur cellulaire à faible fuite

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
FAATZ E. ET AL.: "Cloned structural genes for the osmotically regulated binding - protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12", MOL. MICROBIOL., vol. 2, no. 2, March 1988 (1988-03-01), pages 265 - 279 *
MASUDA J ET AL.: "Transient Tcf3 Gene Repression by TALE-Transcription Factor Targeting", APPL BIOCHEM BIOTECHNOL., vol. 180, no. 8, December 2016 (2016-12-01), pages 1559 - 1573 *

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