CN107344962B

CN107344962B - Repressor protein, regulatory element group, gene expression regulatory system and construction method thereof

Info

Publication number: CN107344962B
Application number: CN201610289098.6A
Authority: CN
Inventors: 娄春波; 侯君然
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2016-05-04
Filing date: 2016-05-04
Publication date: 2021-03-02
Anticipated expiration: 2036-05-04
Also published as: CN107344962A

Abstract

The present invention obtains a repressor protein, a regulatory element group and a gene expression regulatory system having an ultra-sensitive regulatory property by introducing a multimerization domain into the repressor protein and optimizing the number of manipulation sites interacting with the repressor protein in a preferred embodiment.

Description

Repressor protein, regulatory element group, gene expression regulatory system and construction method thereof

Technical Field

The invention belongs to the field of synthetic biology, and relates to an engineered repressor protein, an ultrasensitive regulatory element group, a gene expression regulatory system and construction methods thereof.

Background

Synthetic biology "programs" cells by means of bio-element standardization and conceptual abstraction, allowing artificially designed gene networks to perform a variety of tasks. In recent years, with the rapid development of synthetic biology, the connection between network structure and function is gradually established, and the predictability of gene circuits (circuits) and paths (pathways) is greatly improved, so that the precise and dynamic control of protein nodes, paths and even networks becomes possible.

One of the current research focuses on the use of synthetic biology for dynamic regulation of cellular metabolic pathways in fermentation engineering [1-3 ]. Artificially constructed dynamic regulatory networks, similar to natural networks, often utilize allosteric inhibition or negative feedback loops at the transcriptional level to regulate the expression of key enzymes to alter metabolic flows [4 ]. For example, synthetic networks can be used to modulate the kinetic properties or protein-protein interactions of protein type repressors (i.e., repressor proteins) that respond to exogenous stimuli or endogenous metabolites, allowing optimization of the dynamic regulatory properties of the repressors, and thus, increased metabolite yields. In particular, negative feedback systems comprising repressors enable endogenous metabolites to drive "repair" circuits, addressing metabolic abnormalities in a self-regulating manner to maintain systemic stability [5-6 ]. However, the performance of these synthetic regulatory networks is often unsatisfactory due to problems of leaky expression, too small hill coefficients, etc.

The prior art has engineered repressors to modify the kinetic properties of the native network. As an example, the Bacillus subtilis repressor protein FapR negatively regulates a number of genes involved in fatty acid and phospholipid metabolism, specifically binding to a 17bp DNA sequence, allosterically in response to the concentration of malonyl CoA, an important compound [7-8 ]. When malonyl-coa levels exceed a threshold, the allosteric FapR dissociates from the manipulation site, thereby derepressing transcription. In this manner, the FapR functions as a sensor of fatty acid biosynthesis status. Yields can be improved by engineering FapR-associated transcriptional kinetic networks [4], or by introducing feedback regulation in metabolic pathways [9 ]. For example, Xu et al have utilized the above-described properties of FapR to construct an integrated malonyl-CoA sensor with two-way switching properties. The sensitivity of the sensor to the dynamic response of pGAP-based malonyl-coa can be altered by adding different numbers of tandem manipulation sequences fapO, such that the saturation activities of the promoters are matched when each switch is turned on, and the yield of fatty acids is greatly increased [4 ]. This superior effect demonstrates the potential of engineered repressors in achieving network dynamics, however, this network construction approach relies heavily on the specific properties of FapR and is not universally applicable to engineering repressors.

Furthermore, in order to achieve an ultrasensitive on-off response in gene network dynamic regulation, there is still a need in the art for simple design concepts such as improvements in response efficiency or repressor fold change. Recent studies have controlled background expression by adding corepressor binding sites to enhance the signal-to-noise ratio of the output [10 ]. As a different inventive concept, the present inventors were inspired by the first isolated switch-like transcription repressor, CI protein of lambda phage and LacI protein of bacteria [11-12], and introduced a synergistic effect into the design of regulatory system, thereby enabling the construction of more efficient gene switches.

Disclosure of Invention

In a first aspect, the present invention provides a method of increasing the transcriptional repression capacity of a repressor protein, said method comprising: operably linking a multimerization domain to a DNA-binding domain of the repressor protein, wherein the multimerization domain is selected from the group consisting of multimerization domains of CI family repressor proteins.

In a second aspect, the invention provides a repressor protein comprising a multimerization domain and a DNA binding domain, wherein the multimerization domain is selected from the group consisting of the multimerization domains of CI family repressor proteins.

In a third aspect, the present invention provides a method of increasing the sensitivity of a gene regulatory network, the method comprising:

operably linking a multimerization domain to a repressor protein in the gene regulatory network, wherein the multimerization domain is selected from the group consisting of multimerization domains of CI family repressor proteins; and

the promoter that interacts with the repressor protein is arranged to comprise one or two pairs of operator sites that interact with the DNA binding domain of the repressor protein.

In a fourth aspect, the present invention provides a method of improving the switching properties of a promoter in a host cell, the method comprising:

setting the promoter to comprise one or two manipulation site pairs; and

expressing in the host cell a repressor protein comprising a DNA binding domain capable of binding to the manipulation site and a multimerization domain selected from the group consisting of the multimerization domain of a CI family repressor protein.

In a fifth aspect, the present invention provides a regulatory element group comprising:

a repressor protein comprising a DNA binding domain and a multimerization domain, wherein the multimerization domain is selected from the group consisting of the multimerization domain of a CI family repressor protein; and

a promoter comprising one or two pairs of manipulation sites that interact with the DNA binding domain of the repressor protein.

In a sixth aspect, the present invention provides a gene expression regulation system comprising:

a first gene comprising a sequence encoding a repressor protein for expression of the repressor protein in a host cell, the repressor protein comprising a DNA binding domain and a multimerization domain, wherein the multimerization domain is selected from the group consisting of the multimerization domains of CI family repressor proteins; and

a second gene comprising a sequence encoding a protein to be regulated for expression of the protein to be regulated in a host cell, the second gene further comprising a promoter sequence comprising one or two pairs of manipulation sites that interact with the DNA binding domain.

Advantageous effects

The invention links the multimerization domain of a CI family repressor protein to a native repressor protein without destroying the DNA binding domain of the repressor protein interacting with its target DNA, and thus realizes dimerization, tetramerization and even octamerization of the repressor protein. Since steric hindrance in the vicinity of the promoter sequence interacting with the repressor protein increases after multimerization of the repressor protein, competitive inhibition of RNA polymerase is increased, and the probability of RNA polymerase binding decreases, thus enabling efficient reduction of overexpression. In this way, the transcriptional repression of the repressor protein can be increased. In particular, as demonstrated by the preferred embodiment of the present invention, by optimizing the multimerization domain, in particular using the multimerization domain of a CI family repressor protein, the switching properties of the promoters in the set of regulatory elements can be further improved compared to engineered proteins using a LacI multimerization domain.

In a preferred embodiment, when the promoter region comprises one pair of manipulation sites by means of engineering methods, the repressor protein binds to each manipulation site in a tetramerized form, and the DNA of the promoter region generates a loop (loop) structure. Furthermore, when the promoter region is engineered to contain two pairs of manipulation sites, the two tetramerized repressor proteins can dimerize again to form a repressor octamer, allowing the DNA of the promoter region to form a tighter loop, further increasing the energy required for transcription initiation. The modular approach is almost suitable for improving the switching performance of any natural set of negative regulatory elements. The method of the invention can be used for improving the promoter naturally containing the manipulation site, and can introduce one or two exogenous manipulation site pairs into a constitutive expression promoter by an engineering method, express corresponding repressor protein in a host cell and construct an inducible promoter with excellent switching performance. In this way, the problem of leaky expression of the T7 promoter, which has plagued the long-standing state of the art, can be solved.

In a preferred embodiment, the distance between the manipulation sites is optimized such that the transcription start site of the promoter is located inside the loop after binding of the repressor protein to each manipulation site. Such a topological conformation allows for greater steric hindrance and more efficient repression of transcription driven by the promoter.

Drawings

Fig. 1 is a schematic design diagram of a regulating element group according to an embodiment of the present invention. Among these, engineered repressor proteins, by virtue of having multimerization domains, are capable of binding to a binding site (i.e., an operator site) in multimeric form. Promoter type 0+ 1: a promoter has only one manipulation site located downstream of the transcription initiation site. Depending on the type of repressor protein, the repressor protein binds to the promoter in monomeric or dimeric form to inhibit transcription. 1+1 type promoter: the promoter has a pair of manipulation sites located upstream and downstream of the transcription initiation site, respectively. The repressor protein binds to the promoter in tetrameric form to inhibit transcription. 1+1+1+1 type promoter: the promoter has two pairs of manipulation sites, one pair located upstream of the distal end of the transcription initiation site and the other pair, i.e., the pair in the 1+1 type promoter. The repressor protein binds to the promoter in the form of an octamer to inhibit transcription. Wherein a multimerization domain from a CI family repressor protein is added to a native repressor protein to construct an engineered repressor protein; various regulatable promoters were constructed by adding different numbers of manipulation sites to the T7 promoter. Binding of the repressor protein to the type 1+1 promoter forms a DNA loop. Binding of repressor protein to the 1+1+1+1 type promoter forms a more compact DNA loop.

Fig. 2 shows input/output curves of the respective control element groups. (a) And (3) regulating the input and output curves of the element groups by using PhlF. (b) And each LmrA regulates and controls the input and output curves of the element group. Data points are experimental measurements, error bars show mean ± SD from three independent experiments, solid line is fitting measured data points to hill equation, dashed line is extension of fitting equation. The insert shows the hill coefficient of each curve, and the bar chart in the insert corresponds to the input and output curves marked from top to bottom in the figure respectively from left to right.

Fig. 3 shows input/output curves of the respective control element groups. (a) And (3) adopting input and output curves of CymR regulatory element groups with different oligomerization structural domains. (b) Input and output curves of the CymR regulatory element group with different connecting peptide lengths are adopted. (c) LmrA with different O2-O3 pitches was used to manipulate the input-output curves of the element sets. Data points are experimental measurements and error bars show mean ± SD from three independent experiments.

FIG. 4 shows the input-output curves and changes in Hill coefficient for nine engineered repressor proteins when combined with a type 0+1 or type 1+1 promoter. The set of regulatory elements consisting of the engineered repressor protein and the 1+1 type promoter is shown in black. The set of regulatory elements consisting of the engineered repressor protein and the type 0+1 promoter is shown in grey. The numbers in the upper left corner of each subgraph show the improvement of the Hill coefficient of the input-output curve of the 1+1 type promoter compared with the Hill coefficient of the 0+1 type promoter. Data points are experimental measurements, error bars show mean ± SD from three independent experiments, solid line is fitting measured data points to hill equation, dashed line is extension of fitting equation.

Figure 5 shows the effect of multimerization domains on inducible promoter induction performance. (a) A CymR regulatory element group; (b) the PhlF regulatory element group. The horizontal axis represents the concentration of the inducer, and the vertical axis represents the output. IPTG concentrations indicate the relative amounts of repressor proteins. Data points are experimental measurements, error bars show mean ± SD from three independent experiments, and the solid line is the fit of measured data points to the hill equation.

Detailed Description

It is known that in gene regulatory systems, substances regulating gene transcription control transcription by binding to specific sites on DNA. A system that regulates a target gene in such a manner that the expression activity of the target gene is turned off or reduced by binding of a regulator to a specific site of DNA is called a negative regulatory system. Negative regulatory systems typically comprise an operator/operator site (operator) and a repressor protein (repressor). The operator gene is a DNA fragment capable of binding to a regulatory protein, so that the transcriptional activity of a target gene is controlled. The operator is typically located near, or spatially adjacent to, the transcription initiation site of the target gene (e.g., the operator sequence is further from the promoter core sequence, however when a regulatory protein such as a repressor protein multimerizes, the operator site that binds to the repressor protein multimer is "caught" near the promoter core sequence). Repressor proteins are regulatory proteins encoded by regulatory genes in a negative regulatory system that, when bound to an operator site, inhibit the binding of RNA polymerase to the promoter region, thereby preventing or attenuating transcription of the target gene. In a negative control inducible system, the target gene is transcribed when the repressor protein binds to the inducer. The most typical negative control induction system is the lactose operon. In the absence of lactose, the lactose operon is repressed because the repressor protein lacI binds to the operator site lacO, constituting a competitive inhibition of the RNA polymerase. However, because of the probability of separation of the repressor protein and the operator, several molecules of β -galactosidase and permease may be produced per cell even when the operator is in the off state. When lactose exists in the environment, the lactose is converted into allolactose in cells by a small amount of transenzyme transport and catalysis of beta-galactosidase and acetyl transferase, the allolactose is used as an inducer molecule to be combined with repressor protein, so that the configuration of the repressor protein is changed, the repressor protein is dissociated from lacO sequence, transcription is carried out, and the beta-galactosidase molecule is increased by 1000 times. In this way, structural genes associated with the transport and metabolism of lactose are turned from "off" to "on".

The CI repressor protein of lambda phage is a key regulator for efficient conversion of the lysogenic and lytic pathways of lambda phage. The CI repressor protein consists of two domains with unique structures: the N-terminal domain is a DNA binding domain capable of binding to O_RAnd O_LManipulation site binding [13]And contact with RNA polymerase [14 ]](ii) a The C-terminal domain mediates self-assembly, forming homodimers followed by tetramers or octamers. Due to O_R1And O_R2Allows the CI proteins bound to these two manipulation sites to self-assemble into tetramers, the first CI dimer with O_R1Can promote the binding of the second CI dimer to O_R2In combination with (1). Further, at higher CI concentrations, the CI dimer is able to bind to O_RDownstream O at 2000bp_LO of the manipulation site_L1And O_L2And O with_R1And O_R2And O_L1And O_L2Octamerization of bound CI dimers [15-16]. Such higher order multimerization capability of CIs allows for proximity to each other [17]Or relatively far away [18 ]]The manipulation sites of (a) are coordinately regulated. Modeling of structures [20]And mutation experiments [19]Both indicate that the binding of CI multimers is synergistic.Similarly, LacI-mediated DNA looping also employs multimerization domains similar to CI but smaller, and rationally distributed repressor binding sites [21]. In particular, when the DNA is looped, the promoter is located inside the loop, and the repressor has a stronger inhibitory effect on transcription [22-23 ]]. Multimerization of repressors enables higher repression efficiency, thereby increasing the signal-to-noise ratio of transcription.

A variety of Phage-derived repressors structurally similar to CI repressors have been found in the art, collectively known as CI family repressors (Comparative Molecular Biology of Lambda pharmaceuticals, A. Campbell, Annu. Rev. Microbiol.1994.48: 193-222; Determination of Cell name Selection reduction Phage Lambda Infection,

St-Pierre, 2009, PhD article). The CI family repressor proteins include, for example, CI434, HK022CI, TP901CI, and P22C 2. The amino acid sequences of the multimerization domains of CI, CI434, HK022CI, TP901CI, and P22C2 described above are known in the art as set forth in SEQ ID NOs: 1-SEQ ID NO: 5, respectively.

The present invention constructs engineered repressor proteins by adding the multimerization domain of a CI family repressor protein to the end of the repressor protein, enabling multimerization (e.g., dimerization, tetramerization, and/or octamerization) of the repressor protein. This ability to multimerize is independent of the structure of the repressor protein and does not affect the interaction of the repressor protein with its site of manipulation, and therefore can serve as a modular approach to increase the efficiency of repressor repression. As demonstrated in the examples of the present invention, even if there is only one manipulation site on DNA, the blocking ability against RNA polymerase is more excellent at the same expression amount of repressor protein due to the increase of steric hindrance.

The term "repressor" as used herein has a meaning well known in the art and refers to a protein that recognizes a specific operator site and, when bound to an operator sequence, either prevents the binding of RNA polymerase to the promoter sequence or prevents the RNA polymerase from moving forward along the DNA, thus constituting an inhibition of transcription. As used herein, the terms "operator", "operator site" and "operator sequence" are used interchangeably to refer to a DNA sequence that inhibits transcription when bound to a repressor protein. It is noted that the present invention does not have to be used in the context of an operator. In the case where the promoter controls only one target gene, the regulation can be performed by the repressor protein constructing method of the present invention. In this regard, the methods, regulatory systems and repressor proteins of the invention are equally applicable to prokaryotic and eukaryotic hosts.

A nucleic acid molecule (e.g., DNA) is said to be "capable of expressing" a polypeptide if it comprises a nucleotide sequence that contains transcriptional and translational regulatory information and such sequence is "operably linked" to a nucleotide sequence that encodes the polypeptide. An operable linkage is a linkage that regulates the DNA sequence to which the DNA sequence intended to be expressed is linked in a manner that "allows the gene to be expressed in recoverable amounts as a peptide or antibody moiety". The precise nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the similar arts. See, e.g., Sambrook et al, 1989; ausubel et al, 1987. sup. 1993.

With respect to operably linking the multimerization domain to the DNA-binding domain of the repressor protein, the multimerization domain may be linked directly to the N-or C-terminus of the repressor protein, or using a linking peptide to the N-or C-terminus of the repressor protein, as long as the binding of the repressor protein to the operator is not disrupted. The linker peptide is flexible and does not comprise any active domains, preferably consisting of glycine and/or serine. As demonstrated by the examples of the invention, the length of the linker peptide is in fact extremely limited in its effect on the engineered repressor protein and can be of any length. In one embodiment, the linker peptide has the sequence GGGGSGGGGS (SEQ ID NO: 6).

The DNA binding domain is derived from a native or artificially modified repressor protein and is capable of binding to DNA and performing a function of preventing transcription. As described above, the multimerization domain is capable of allowing self-assembly of multiple repressors (e.g., repressors) resulting in synergy.

In some embodiments, the multimerization domain and the DNA-binding domain may be linked using a linking peptide. The choice of linker peptide is described elsewhere herein.

setting the promoter to comprise one or two manipulation site pairs; and

The term gene regulation network as used herein has a meaning well known in the art and refers to a mechanism for controlling gene expression in an organism. The sensitivity of a gene regulatory network is defined as the response to a change in inducer concentration. For ultrasensitive networks, promoters have the ability to respond rapidly to changes in the concentration of the inducer over a narrow range. Small changes in inducer concentration above the threshold value rapidly activate the transcription pathway, and therefore the response curve often resembles a typical step-like response, i.e., exhibits "on-off" characteristics (Achimescu S, Lipan O.Signal amplification in nonlinear stored gene regulation networks. systems Biology, IEE Proceedings, 2006, 153 (3): 120-) -134). Since the induced response curve is often inverse sigmoid, it is usually fitted to a hill equation (e.g., in the form of equation 5 of the examples section). The larger the Hill coefficient, the smaller the hypersensitivity interval, the higher the sensitivity, which indicates the better "switch" performance of the network.

As described above, by making the promoter of the gene to be regulated contain one or two pairs of manipulation sites, the repressor protein can bind to the promoter sequence in the form of a tetramer or octamer, inhibiting the binding of RNA polymerase to the promoter. In a preferred embodiment, the spacing of the pairs of manipulation sites in the prokaryotic host is optimized for the multimerization domain of the CI family repressor protein; however, it should be noted that although the structure of the core promoter of eukaryotes differs from that of prokaryotes, the length of the promoter also differs, however, by adjusting the position of the manipulation site, multimerization of the repressor protein in the promoter region can still be achieved.

In some embodiments, a promoter that interacts with a repressor, such as a repressor protein, is provided that comprises a pair of operator sites that interact with the DNA binding domain of the repressor protein. The pair of manipulation sites may be located both upstream and downstream of the transcription initiation site, or on both sides of the transcription initiation site, respectively. Preferably, the pair of manipulation sites are located on both sides of the transcription initiation site, respectively. Preferably, the separation between the two of said pair of manipulation sites is such that when said repressor protein forms a loop upon binding to said pair of manipulation sites in tetrameric form, said pair of manipulation sites is located ipsilaterally to said loop. Preferably, the separation between two of the pair of manipulation sites is 40-70bp, preferably 50-70 bp. In this way, the repressor protein binds to the promoter in a tetrameric form.

In some embodiments, a promoter that interacts with a repressor, such as a repressor protein, is provided that comprises two pairs of operator sites that interact with the DNA binding domain of the repressor protein. Wherein one manipulation site pair is located upstream or downstream of the transcription initiation site of the promoter or on both sides of the transcription initiation site of the promoter, respectively; the other pair of manipulation sites are both located upstream or downstream of the transcription start site of the promoter. Preferably, one pair of manipulation sites is located on both sides of the transcription initiation site of the promoter, respectively, and the other pair of manipulation sites is located upstream or downstream of the transcription initiation site of the promoter. Preferably, the separation between the two manipulation sites in each pair of manipulation sites is 40-70bp, preferably 50-70 bp. The separation between the two manipulation site pairs is greater than 150bp, preferably greater than 200 bp. In the case of a spacing of more than 150bp, since a particularly tight loop has been formed, there is no need to take into account the conformation around the promoter sequence, nor the length of the spacing. The sequence inserted to complement the number of spacer bases is not particularly limited as long as it has no promoter activity, and may be a randomly generated sequence, for example. In this way, the repressor protein binds to the promoter in the form of an octamer.

The term "promoter" refers to a region of DNA that initiates transcription of a gene, upstream of the synonymous strand from the start of transcription of the gene. In the case of prokaryotes, promoters generally comprise transcriptional regulatory regions (e.g., inducible promoters), RNA polymerase recognition regions, and a transcription initiation site. The term "minimal promoter" refers to the smallest transcriptional control unit capable of initiating transcription, such as a promoter core region that contains only the RNA polymerase recognition region and the transcription initiation site. In the present invention, an inducible promoter excellent in switching performance is constructed by adding one manipulation site, one manipulation site pair or two manipulation site pairs to the upstream and/or downstream of a minimal promoter. The position of the manipulation site in the chimeric promoter thus constructed is as described above.

Embodiments of the aspects described herein may be illustrated by the following numbered paragraphs:

1. a method of increasing the transcriptional repression ability of a repressor protein, the method comprising: operably linking a multimerization domain to a DNA-binding domain of the repressor protein, wherein the multimerization domain is selected from the group consisting of multimerization domains of CI family repressor proteins.

2. The method of paragraph 1, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

3. The method of paragraph 2, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

4. The method of any of paragraphs 1-3, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

5. The method of any of paragraphs 1-4, wherein the multimerization domain is directly linked to the DNA-binding domain.

6. The method of any of paragraphs 1-4, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

7. The method of paragraph 6 wherein the linker peptide is 1-20 amino acids in length, preferably having the amino acid sequence of SEQ ID NO: 6.

8. A repressor protein comprising a multimerization domain and a DNA binding domain, wherein the multimerization domain is selected from the group consisting of the multimerization domains of CI family repressor proteins.

9. The repressor protein of paragraph 8, wherein the DNA binding domain is derived from a native or artificially modified repressor protein.

10. The repressor protein of paragraph 8 or 9, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

11. The repressor protein of paragraph 10, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

12. The repressor protein of any of paragraphs 8 to 11, wherein the multimerization domain is operably linked to the C-terminus of the DNA binding domain.

13. The repressor protein of any of paragraphs 8 to 12, wherein the multimerization domain is directly linked to the DNA binding domain.

14. Repressor according to any of paragraphs 8 to 12, wherein the multimerisation domain is linked to the DNA-binding domain using a linking peptide.

15. The repressor protein of paragraph 14, wherein the connecting peptide is 1 to 20 amino acids in length, preferably having the amino acid sequence of SEQ ID NO: 6.

16. A method of increasing the sensitivity of a gene regulatory network, the method comprising:

17. A method of improving the switching properties of a promoter in a host cell, the method comprising:

setting the promoter to comprise one or two manipulation site pairs; and

18. The method of paragraph 17 wherein said promoter is the T7 promoter.

19. The method of any one of paragraphs 16-18, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

20. The method of paragraph 19, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

21. The method of any of paragraphs 16-20, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

22. The method of any of paragraphs 16-21, wherein the multimerization domain is directly linked to the DNA-binding domain.

23. The method of any of paragraphs 16-21, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

24. The method of paragraph 23 wherein the linker peptide is 1-20 amino acids in length, preferably having the amino acid sequence of SEQ ID NO: 6.

25. The method of any one of paragraphs 16-24, wherein the promoter comprises a pair of manipulation sites, wherein the pair of manipulation sites is located either upstream or downstream, or on both sides, of the transcription start site of the promoter; preferably, the pair of manipulation sites flank the transcription initiation site of the promoter, respectively.

26. The method of paragraph 25, wherein the separation between the two of the pair of manipulation sites is such that when the repressor protein forms a loop upon binding to the pair of manipulation sites in tetrameric form, the pair of manipulation sites is on the same side of the loop.

27. The method of paragraph 25, wherein the separation between the two of the pair of manipulation sites is 40-70bp, preferably 50-70 bp.

28. The method of any one of paragraphs 16-24, wherein the promoter comprises two pairs of manipulation sites, wherein a first pair of manipulation sites is located either upstream or downstream, or on both sides, respectively, of the transcription start site of the promoter, and a second pair of manipulation sites is located either upstream or downstream, or both sides, of the transcription start site of the promoter; preferably, the first pair of manipulation sites are located on either side of the transcription start site of said promoter, respectively, and the second pair of manipulation sites are located both upstream and downstream of the transcription start site of said promoter.

29. The method of paragraph 28, wherein the separation between the two manipulation sites in each pair of manipulation sites is 40-70bp, preferably 50-70 bp.

30. The method of paragraph 29, wherein the spacing between the first pair of manipulation bit points and the second pair of manipulation bit points is greater than 150bp, preferably greater than 200 bp.

31. A set of regulatory elements, comprising:

32. The set of regulatory elements of paragraph 31, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

33. The set of regulatory elements of paragraph 32, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

34. The set of regulatory elements of any of paragraphs 31-33, wherein said multimerization domain is operably linked to the C-terminus of said DNA-binding domain.

35. The set of regulatory elements of any of paragraphs 31-34, wherein said multimerization domain is directly linked to said DNA-binding domain.

36. The set of regulatory elements of any of paragraphs 31-34, wherein said multimerization domain is linked to said DNA-binding domain using a linking peptide.

37. The set of regulatory elements of paragraph 36, wherein the linker peptide is 1-20 amino acids in length, preferably having the amino acid sequence of SEQ ID NO: 6.

38. The set of regulatory elements of any of paragraphs 31-37, wherein said promoter comprises a pair of manipulation sites, wherein said pair of manipulation sites is located either upstream or downstream, or on each side of the transcription start site of said promoter; preferably, the pair of manipulation sites flank the transcription initiation site of the promoter, respectively.

39. The set of regulatory elements of paragraph 38, wherein the separation between the two of the pair of manipulation sites is such that when the repressor protein forms a loop upon binding to the pair of manipulation sites in a tetrameric form, the pair of manipulation sites is on the same side of the loop.

40. The set of regulatory elements of paragraph 38, wherein the pair of manipulation sites flank the transcription initiation site of the promoter; preferably, the separation between two of the pair of manipulation sites is 40-70bp, preferably 50-70 bp.

41. The set of regulatory elements of any of paragraphs 31-37, wherein said promoter comprises two pairs of manipulation sites, wherein a first pair of manipulation sites is located either upstream or downstream, or on both sides, of the transcription start site of said promoter, and a second pair of manipulation sites is located either upstream or downstream, respectively, of the transcription start site of said promoter; preferably, the first pair of manipulation sites are located on either side of the transcription start site of said promoter, respectively, and the second pair of manipulation sites are located both upstream and downstream of the transcription start site of said promoter.

42. The set of regulatory elements according to paragraph 41, wherein the separation between the two manipulation sites in each pair of manipulation sites is 40-70bp, preferably 50-70 bp.

43. The set of regulatory elements of paragraph 42, wherein the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150bp, preferably greater than 200 bp.

44. A gene expression regulation system, the system comprising:

45. The system of paragraph 44 wherein the second gene is a gene associated with a metabolic pathway.

46. The system of paragraphs 44 or 45 wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

47. The system of paragraph 46, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

48. The system of any of paragraphs 44-47, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

49. The system of any of paragraphs 44-48, wherein the multimerization domain is directly linked to the DNA-binding domain.

50. The system of any of paragraphs 44-48, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

51. The system of paragraph 50, wherein the linker peptide is 1-20 amino acids in length, preferably having the amino acid sequence of SEQ ID NO: 6.

52. The system of any one of paragraphs 44-51, wherein the promoter comprises a pair of manipulation sites, wherein the pair of manipulation sites is located either upstream or downstream, or on both sides, of the transcription start site of the promoter; preferably, the pair of manipulation sites flank the transcription initiation site of the promoter, respectively.

53. The system of paragraph 52, wherein the separation between the two of the pair of manipulation sites is such that when the repressor protein forms a loop upon binding to the pair of manipulation sites in tetrameric form, the pair of manipulation sites is on the same side of the loop.

54. The system of paragraph 52, wherein the pair of manipulation sites flank the transcription start site of the promoter, respectively; preferably, the separation between two of the pair of manipulation sites is 40-70bp, preferably 50-70 bp.

55. The system of any one of paragraphs 44-51, wherein the promoter comprises two pairs of manipulation sites, wherein a first pair of manipulation sites is located either upstream or downstream, or on both sides, respectively, of the transcription start site of the promoter, and a second pair of manipulation sites is located either upstream or downstream, or both sides, of the transcription start site of the promoter; preferably, the first pair of manipulation sites are located on either side of the transcription start site of said promoter, respectively, and the second pair of manipulation sites are located both upstream and downstream of the transcription start site of said promoter.

56. The system of paragraph 55 wherein the separation between the two manipulation sites in each pair of manipulation sites is 40-70bp, preferably 50-70 bp.

57. The system of paragraph 56 wherein the spacing between the first pair of manipulation bit-points and the second pair of manipulation bit-points is greater than 150bp, preferably greater than 200 bp.

Examples

The examples of the present invention engineered 9 completely different wild-type repressors and their manipulation sites known in the prior art, prepared ultrasensitive transcription control elements with different transcription control strengths, and demonstrated the effects obtained by adding multimerization domains to repressors and/or manipulation sites in promoter regions. The wild-type repressor protein and its operating site are shown in Table 1.

TABLE 1 wild-type repressor protein and its corresponding operator site sequence

1. Engineering of repressor proteins

The CI multimerization domain (corresponding to amino acids 136-236 of the CI protein, the DNA sequence encoding this domain being SEQ ID NO: 25) or the CI434 multimerization domain (corresponding to amino acids 70-210 of the CI434 protein, the DNA sequence encoding this domain being SEQ ID NO: 26) was fused to the wild-type repressor protein shown in Table 1. Unless otherwise indicated, repressor proteins capable of multimerization were constructed by linking the two using a flexible short linker peptide (corresponding coding sequence of SEQ ID NO: 27). As a control, a LacI multimerization domain (corresponding to amino acid 341-360 of LacI protein, the DNA sequence encoding this domain is SEQ ID NO: 28) was fused to the wild-type repressor protein and a flexible short linker peptide (corresponding to encoding sequence SEQ ID NO: 27) was used to join the two to construct a repressor protein capable of multimerization. In each figure, the wild-type repressor protein is denoted by "-wt", the engineered repressor protein with the CI multimerization domain added to the C-terminus is denoted by "-CI" and the engineered repressor protein with the CI434 multimerization domain added to the C-terminus is denoted by "-CI 434", and the engineered repressor protein with the LacI multimerization domain added to the C-terminus is denoted by "-LacI".

2. Construction of promoters with different manipulation sites

As shown in FIG. 1, the following four promoters were constructed by adding manipulation sites upstream and/or downstream of the core region (i.e., core promoter or minimal promoter, SEQ ID NO: 29) of the wild type promoter of T7:

the wild-type T7 promoter;

promoter type 0+ 1: the promoter region presents only one manipulation site downstream of the transcription start site;

1+1 type promoter: the promoter region has an operation site pair which is respectively positioned at the upstream and downstream of the transcription initiation site;

1+1+1+1 type promoter: two pairs of manipulation sites are present in the promoter region, one pair being located both upstream and the other pair being located respectively upstream and downstream of the transcription start site.

In addition, T7RNA polymerase is integrated into the cell such that the cell expresses sufficient levels of T7RNA polymerase.

For the 0+1 type promoter, the manipulation site (O1) is located downstream of the minimal promoter and is contiguous with the T7 minimal promoter.

In the case of the 1+1 type promoter, in order to ensure that two dimerization repressors are respectively bound to mutually separated ipsilateral sites of a DNA helix to make a loop structure, the distance between the repressor binding sites of the 1+1 type promoter is set to about 4 to 6 DNA pitches. Early studies found that CI proteins bind synergistically to a pair of manipulation sites separated by 5 or 6 helices [24 ]. Furthermore, Becker et al found that the activity of the reporter is periodic when the promoter is made to move by several bp between two manipulation sites, indicating that stronger repression can be achieved when the promoter is on the inner surface of an unfavorably tight DNA loop [23 ]. In this regard, we placed one manipulation site (O1) downstream of the T7 minimal promoter and contiguous with the T7 minimal promoter, and another manipulation site (O2) upstream of the promoter, with a center-to-center spacing of O1 to O2 of about 40-70bp (see Table 2 in particular). In this way, it is ensured that the repressor protein competes with T7RNA polymerase in both directions and that the steric hindrance of the loop at the promoter is more supported towards the inside of the DNA loop. It is noted that the sequences added between the O2 and T7 minimal promoters are random sequences for occupancy purposes only, and no promoter activity was predicted to be verified by software.

With respect to promoters of the 1+1+1+1 type, we would like to further improve the switching performance of the regulatory element set by adding more manipulation sites upstream of the promoter, to achieve higher order multimerization (e.g., octamerization) of the engineered repressor protein, taking into account that the native CI repressor protein is able to form octamers and thus form tighter DNA loops [19 ]. The 1+1+1+1 type promoter includes 4 identical repressor protein binding sites (i.e., operator sequences). Wherein the first manipulation site (O1) and the second manipulation site (O2) are arranged as same as the 1+1 type promoter, and the center distance between the third manipulation site (O3) and the fourth manipulation site (O4) is 50 bp. The length of the random sequence between O2 and O3 was set to about 200bp (198bp), unless otherwise indicated.

The type 0+1 and type 1+1 promoters constructed in the examples are shown in Table 2. Among them, the bold part is an operator sequence (i.e., SEQ ID NO: 16-SEQ ID NO: 24 of Table 1), the italic part is a T7 core promoter (i.e., SEQ ID NO: 29), and the other (lower case letters) is a random sequence for maintaining the interval of operator sites.

TABLE 2 engineered promoters

3. Construction of measurement systems

In order to measure the input (repressor) -output (promoter) response curve, sfGFP (corresponding to the coding sequence SEQ ID NO: 30) was used as a reporter to be ligated downstream of the promoter constructed in the present invention, and the fluorescence amount of sfGFP was used as an output signal of each promoter. On the other hand, wild-type or engineered LmrA, PhlF, AmtR, YefM, CymR, MazE, Cro, FapR, TrpR repressor protein under the control of the Ptac promoter (with lower leakage expression) and constitutively overexpressed LacI were inserted into the same RGP low copy plasmid (SEQ ID NO: 31) (see materials and methods section below for details). In this manner, IPTG induction at various concentrations is used to control the amount of expression of the wild-type or engineered repressor protein.

4. Effect of multimerization on regulatory Performance

To investigate whether multimerization can enhance repression performance, we measured input-output response curves for different combinations: wild-type repressor/engineered repressor in different combinations with 0+1 type promoter (repressor monomeric or dimeric), 1+1 type promoter (repressor tetrameric), or 1+1+1 type promoter (repressor octamer). As shown in fig. 2(a), the performance of the regulatory element group consisting of the 1+1 type promoter and the wild-type PhlF repressor protein (hill coefficient of 1.77) was almost completely consistent with the performance of the regulatory element group consisting of the 0+1 type promoter and the wild-type PhlF repressor protein (hill coefficient of 1.93). This suggests that O2 exerts little regulatory effect in the absence of the multimerization domain, even though the repressor protein is able to bind at two separate manipulation sites, due to the lack of synergy and some distance from the core promoter. In contrast, the switching performance of the set of regulatory elements consisting of a type 1+1 promoter and an engineered PhlF repressor protein with the addition of a CI434 multimerization domain (hill coefficient of 3.42) was significantly improved compared to the set of regulatory elements consisting of a type 0+1 promoter and an engineered PhlF repressor protein with the addition of a CI434 multimerization domain (hill coefficient of 2.18). This indicates that the upstream and downstream binding sites of the type 1+1 promoter allow the formation of PhlF tetramers, which are more resistant to T7RNA polymerase binding to the promoter.

As another example, as shown in figure 2(b), the engineered LmrA repressor protein shows the same trend as the engineered PhlF when used in combination with a type 0+1 promoter or with a type 1+1 promoter (hill coefficients of 1.44 and 3.89, respectively). It is noted that the engineered LmrA repressor complexed with the promoter type 0+1 (with a Hill coefficient of 1.44) is more repressible than the wild-type LmrA repressor complexed with the promoter type 0+1 (with a Hill coefficient of 0.83), probably because the multimerization domain of the engineered repressor recruits more repressor and thus the local concentration of repressor increases, resulting in greater steric resistance. In this regard, the improved performance of the engineered regulatory element set can be attributed to the following two points: (1) DNA cyclization by tetramerization of each repressor protein on the 1+ 1-type promoter; and (2) the multimerization domain itself attracts more of the repressor protein, so that the repressor protein is enriched locally.

To quantify this improvement in repression, we fit the data using the least squares method with repressive hill equations as models (see methods section below) and quantitatively characterize the regulatory properties of these constructs using hill coefficients (expressed as n). The higher the hill coefficient, the steeper the induction curve due to synergy, indicating that the regulatory element set is more sensitive. For example, as shown in the inset of fig. 2(b), the hill coefficient for the LmrA-CI434 repressor protein when it is incorporated with the 0+1 type promoter (dimerization) is 1.44, the hill coefficient when it is incorporated with the 1+1 type promoter (tetramerization) is 3.89, and the hill coefficient when it is incorporated with the 1+1+1 type promoter (octamer) is 5.19. This indicates that sensitivity can be improved by 73% by increasing steric hindrance only by multimerization, compared to wild-type (monomer) complexed 0+ 1-type promoter; sensitivity can be increased by 170% again by tetramerisation of the repressor protein; while sensitivity can be further increased by 33% by octamerizing the repressor protein. These results demonstrate that the CI434 multimerization domain multimerizes the repressor protein, yet it does not alter the specific binding affinity between the repressor protein and the DNA and the conformation of the DNA binding domain.

It can be seen that when the set of regulatory elements has 2 or 4 manipulation sites, the repressor protein is capable of higher order multimerization (tetramerization or octamerization) compared to dimerization of the repressor protein or binding of the monomer to a single promoter, thereby enabling the creation of a loop structure in the promoter region. Such an arrangement allows the promoter to be turned off more tightly. Also, because of the synergy of multimerization, the dose response curve for such constructs is steeper, with a larger hill coefficient, indicating better sensitivity of the promoter.

5. Effect of the type of multimerization Domain on the sensitivity of the dose response Curve

As shown in fig. 3(a), for the engineered CymR, the repressor protein with both CI and CI434 multimerization domains showed significant hypersensitivity as compared to the wild-type CymR. However, the performance of CymR fused with a multimerization domain from LacI is not satisfactory: the performance of the CymR-lacI 0+1 and CymR-lacI 1+1 regulatory element groups is almost not different, which indicates that the repressor protein fused with the LacI multimerization structural domain can not realize good tetramerization under the condition. This is hypothesized to be due to the fact that the 20 amino acid multimerization domain located at the C-terminus of LacI is small, inherently less multimerizing, or otherwise susceptible to the spatial structure of the repressor protein being fused and unable to function as a multimerization.

6. Effect of linker peptides on the Performance of engineered repressor proteins

The DNA binding domain and multimerization domain of the native CI or CI434 protein each have a short flexible linker peptide (about 40aa) between them, and proteolytic cleavage of the linker peptide disrupts the ability to form tetramers [15 ]]. In view of this, we investigated the effect of linker peptide length on repression performance. Fusion repressor proteins are constructed by linking the CI434 multimerization domain to the C-terminus of the native CymR repressor protein using three lengths of linker peptides, two consecutive GGGGS linker peptides (GGGGS)₂(10aa), a single GGGGS linker peptide (5aa) and no linker peptide. As shown in fig. 3(b), lengthening of the flexible linker peptide only slightly improved repression performance, where (GS) corresponds to a single GGGGS linker peptide; (GS)₂Corresponding to two consecutive GGGGS connecting peptides (GGGGS)₂. In addition, the engineered repressor protein is capable of tetramerization even without a linker peptide. It can be said that, in the case of the present invention, (GGGGS)₂Length and flexibility ofIt is sufficient for achieving the effects of the present invention.

7. Effect of distal steering site spacing on steering Performance

As described above, the addition of distal manipulation sites O3 and O4 enables the promoter region to form an octamer repressor protein with more complex DNA loops. To investigate the relationship between the set position of the distal steering site and the regulatory performance, the spacer sequence between O2 and O3 was set to three lengths (about 200, about 500, and about 700 bp). The spacer sequence is a random sequence predicted to demonstrate no promoter activity. Wherein the about 700bp random sequence (actual length 692bp) is found in SEQ ID NO: 32, a first step of removing the first layer; the random sequence of about 500bp (actual length 522bp) is SEQ ID NO: 32 from nucleotide 1 to nucleotide 522; the random sequence of about 200bp (actual length 198bp) is SEQ ID NO: 32 from nucleotide 1 to nucleotide 198. It is to be noted that the topology is also less controllable due to the longer spacer sequence, in which case it is not necessary to consider whether the core promoter is located inside or outside the loop. As shown in FIG. 3(c), changing the distance between the long-distance promoter O3/O4 and O1/O2 did not have much influence on the induction curve. In fact, better input-output response is achieved when the spacings of O2 and O3 are about 200, 500, and 700bp, respectively.

8. Universality verification of the method of the invention

For the nine engineered repressors constructed in this example, promoters of type 0+1 or 1+1 containing their manipulation sites were constructed, respectively. As shown in FIG. 4, for the nine different repressor proteins employed, the sensitivity of the regulatory element set can be increased by allowing multimerization of the repressor proteins and introducing multiple manipulation sites in the promoter region. Specifically, tetramerization near the core promoter increased the hill coefficient of the regulatory element set by 92% -218% compared to dimerization.

9. Effect of multimerization Domain on inducible promoter Induction Performance

Promoters in the wild-type PhlF regulatory element set and the CymR regulatory element set can be induced by DAPG and cumate, respectively. To determine whether the multimerization domain has an effect on the inducible performance of inducible promoters, we studied the inducible performance of the regulatory element sets of engineered CymR and PhlF repressor proteins with the addition of CI434 multimerization domain and wild-type repressor protein in combination with 1+ 1-type promoter, respectively. To allow comparison of the magnitude of the induced properties, we used different IPTG concentrations to control the wild-type or engineered repressor protein. As shown in fig. 5, the inducibility of both inducible regulatory elements is not affected by the multimerization domain: CymR-CI434 can still be induced by cumate; PhlF-CI434 was still inducible by DAPG. Furthermore, in the case of the 1+1 type promoter, the IPTG concentration used was greatly reduced, indicating that the engineered regulatory element set was able to respond to a smaller concentration of repressor protein, indicating that both CymR-CI434 and Phlf-CI434 respond more sensitively to the inducer than the wild-type protein.

10. Conclusion

In this example, we show a modular approach to effective improvement of repressor performance. The method is widely applicable to various repressor proteins. In particular, by introducing a DNA loop in the promoter region by making the promoter region contain multiple manipulation sites, the switching performance or sensitivity of the promoter can be further improved. As an example, inactivated repressor protein (MazE) in a toxicity-antitoxic system is rescued; by fusing the multimerization module to the antitoxic protein in the absence of the toxic protein, it can act as a normal, or stronger, repressor protein. Our design approach can greatly improve the flexibility of metabolic dynamics control and provide a kit of regulatory elements with ultra-high sensitivity repression for synthetic biology.

In addition, as is known in the art, the expression of the T7 promoter is very strong, which is extremely difficult to "turn off. Therefore, our method also enables the construction of a regulatable T7 promoter with good switching performance.

11. Materials and methods

11.1 reagents and media

Luria-Bertani (LB) liquid Medium:

peptone (Fisher Scientific) 10 g/l;

NaCl(Fisher Scientific) 10g/l；

yeast powder (Fisher Scientific) 5 g/l.

Autoclaving at 121 deg.C for 20 min.

LB solid medium:

the formula of the liquid culture medium is the same as that of the LB liquid culture medium, and 1.5 percent of agar powder is added.

M9 minimal medium supplemented with salts:

chloramphenicol (Acros): dissolved in LB or M9 medium to a final concentration of 20. mu.g/ml.

Ampicillin (Acros): dissolved in LB or M9 medium to a final concentration of 25. mu.g/ml.

Isopropyl-. beta. -D-1-thiogalactoside (IPTG) (USB Corporation) was stored at 1M, and the final concentrations were 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 50. mu.M, 70. mu.M, 100. mu.M, 200. mu.M, 500. mu.M, 800. mu.M and 1500. mu.M, respectively.

The stock concentration of L- (+) -arabinose (sigma) was 1M, and the final concentrations were 0.1mM, 1mM, 2mM and 5mM, respectively.

4-isopropylbenzoic acid (cumate) (J & K) stock solution was 1M at final concentrations of 0.1. mu.M, 0.2. mu.M, 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M, 100. mu.M and 1000. mu.M, respectively.

The stock solution of 2, 4-Diacetylphloroglucinol (DAPG) (ChemCruz) was at a concentration of 200mM, with final concentrations of 0.01. mu.M, 0.05. mu.M, 0.2. mu.M, 0.5. mu.M, 1. mu.M, 2. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 50. mu.M and 100. mu.M, respectively.

PBS buffer:

NaCl: 7.9g (Beijing chemical plant, purity is more than or equal to 99.5%);

KCl: 0.2g (Beijing chemical plant, purity is more than or equal to 99.5%);

KH₂PO₄: 0.24g (Beijing chemical plant, purity is more than or equal to 99.5%);

K₂HPO₄: 1.8g (Beijing chemical plant, purity is more than or equal to 99.0%),

dissolving in 800ml distilled water, adding distilled water to constant volume to 1L, and adjusting pH to 7.4 with HCl (analytically pure, chemical reagent of national pharmaceutical group, Ltd.).

PBS buffer supplemented with 2mg/ml kanamycin.

LB liquid and solid media were used in plasmid construction and strain maintenance. The strains were cultured in medium, and M9 minimal medium supplemented with salts was used for induction.

Restriction enzymes, T4 polynucleotide kinase and T4DNA ligase were purchased from New England Biolabs (Frankfurt, Germany). Unless otherwise stated, use OLMA: (A)oligo-linker mediated assembly) method [25]A plasmid was constructed. Primers and single-stranded DNA fragments (oligonucleotides) were purchased from BGI (beijing genomics). Using Nano

The DNA concentration was measured with a spectrophotometer ND-2000(Peqlab, Erlangen, Germany).

11.2 strains and plasmids

All plasmid constructions used E.coli strain DH5 alpha (TransGen Biotech) as cloning strain. As a test strain, Escherichia coli T7E1a strain [25] which is a DH10B (TransGen Biotech) strain integrating T7RNA polymerase transcription units into bacterial chromosomes using pOSIP plasmid was used.

Programmed DNA assembly was performed for the RGP series plasmid (SEQ ID NO: 31) and the PTP series plasmid (SEQ ID NO: 33). RGPs incorporate different repressors driven by the Lac promoter, and in addition, except for the region of p15A, the plasmid incorporates constitutively overexpressed LacI and AmpR; in addition, two Bsal sites flank the lacZ α fragment, so that the recognition site is removed from the plasmid after digestion. The RGP series plasmids were constructed by replacing the LacZ α fragment with the wild-type or engineered repressor protein of interest using the Golden Gate cloning method. PTPs were prepared to characterize the induction strength of the different promoters. In addition to pSC101 and CmR, PTP also have two Bsal sites flanking the lacZ α fragment, so that the promoter of interest is inserted by digestion in place of the lacZ α fragment. Promoter DNA fragments were prepared by chemical synthesis and ligated into each plasmid.

RGP plasmid and PTP empty plasmid (same as PTP resistant copy number, no GFP transcription unit) expressing repressor protein were transferred into E.coli T7E1a strain as negative control, and fluorescence value of the negative control was subtracted as background in flow cytometry data.

11.3 cell growth

According to [26], the cells are cultured before the fluorescence measurement. First, monoclonal bacteria were selected on LB plates, inoculated into 4ml of LB in a Falcon tube, and cultured overnight on a shaker at 37 ℃ and 250 rpm. The overnight cultures were diluted 1:200 to 96-well plates using pre-warmed M9 medium. Incubate at 37 ℃ in a high speed orbital shaking Safire microplate spectrophotometer (Tecan) and record OD600 measurements for each well every 5 min. Once the OD600 of the diluted culture reached 0.12-0.14 (-3 h), the culture was diluted 700-fold to a new 96-well plate using pre-warmed M9 medium containing IPTG. Exponential growth was maintained by induction in a digital readout thermostated shaker (Elmi) at 1,000rpm for 6 hours. Finally, each culture was transferred to a new plate containing 150. mu.l PBS and 2mg/ml kanamycin to terminate protein expression.

11.4 fluorescence assay

The fluorescence distribution of each sample was measured using a LSRII flow cytometer (BD Biosciences) with the voltages set at FSC:516, SSC:286, FITC:680, B: 650. Each measurement comprises at least 50,000 sample volumes. Using FlowJo (v7.6), gates were set based on forward scatter and side scatter. The geometric mean of each sample was calculated. The fluorescence measurement of E.coli DH10B cells containing only the negative control plasmid was used as autofluorescence, and the average value was corrected.

11.5 data analysis and modeling

All data fits were performed using MATLAB 7.11.0.584 version (R2010b) (Mathworks) and plotted using MATLAB and Graph Pad Prism 5.

For modeling gene expression in cells, models of different scales focusing on different effector molecules have been reported in the art. Among the key variables that are adjustable at the transcription level include the copy number acting on the gene of interest, the number of transcription factors, the strength of transcription factor binding, the strength of RNA polymerase binding, and the like; at the translational level, key variables that can be adjusted include the strength of the ribosome binding site, the rate of degradation of the protein product to which the gene of interest corresponds, and the like. For the purposes of the present invention to enhance induction by increasing transcription factor binding sites, the other parameters are constants except for the following two parameters: when the transcriptional regulation is simply repressed (i.e., there is only one transcription factor binding site), only the number of transcription factors need to be considered; when a plurality of transcription factor binding sites which may exert a synergistic effect are present on the promoter, the number of transcription factors and the strength of the synergistic effect need to be considered. The invention adopts a transcription thermodynamic model based on Rob Philips and the like. The rationale is that transcriptional activity is proportional to the probability of observed RNA polymerase (RNAP) binding to the promoter; also, the gene expression level (i.e., the fluorescence intensity measured in the experiment) was approximated to the transcription level. For only one repressor binding site and multiple repressor binding sites present upstream and downstream of the T7 minimal promoter, the probability that RNAP binds to the promoter is:

wherein, P and R are the number of RNA polymerase and repressor protein, respectively; k_pdAnd K_O1Dissociation constants for RNAP dissociation from the T7 promoter and for dissociation of a single repressor from the repressor binding site in vivo, respectively; n is the Hill coefficient, characterizing the synergistic effect of a repressor protein binding to multiple repressor protein binding sites to form dimers or higher order oligomers. Due to P/K_pdIs constant and P/K_pd>>1, equation (1) can be simplified to

During the exponential growth phase of the cells, the dynamic changes in protein concentration can be described using ordinary differential equations:

where G is the concentration of the protein of interest, and in the measurements of the invention G is the concentration of sfGFP. Previous measurements indicated that the amount of fluorescence per cell in exponential growth phase was a solution where dG/dt is 0. That is to say that the first and second electrodes,

as discussed above, the reduction/dilution of proteins in bacteria is generally caused by dilution resulting from cell division and degradation of the proteins themselves. However, for most proteins, in particular sfGFP measured here, the degradation rate is several orders of magnitude (hundreds of times) smaller than the dilution rate, and therefore the degradation rate depends only on the rate of the logarithmic growth phase of the cells (in the case of bacteria, γ ≈ 1/hour), and therefore the measured GFP level is proportional to β + (V)_max-β)·P_boundI.e. by

The fluorescence level is related to R by an inverse sigmoidal curve. The results of the experiment were compared with fluorescence measurements, and Vmax is the expression level of the T7 minimal promoter without transcription factor binding site, that is to say the expression level in the case of R ═ 0. In the experiments of the present invention, Vmax was 2388 (a.u.). Beta is a parameter that depends on the strength of the binding site for the repressor protein.

The data were fitted to equation (5) using the least squares method, where K and n are free variables. The least squares method is performed using fminsearch function of matlab, and parameters are determined by searching the minimum of the sum of each data set. In this way, the placement of each repressor protein and its binding site is quantitatively characterized and the hill coefficient n is obtained.

11.6 reference

1.Oyarzun DA,Stan GB:Synthetic gene circuits for metabolic control:design trade-offs and constraints.Journal of the Royal Society,Interface/the Royal Society 2012.

2.Nielsen J,Keasling JD:Synergies between synthetic biology and metabolic engineering.Nature biotechnology 2011,29(8):693-695.

3.Boyle PM,Silver PA:Parts plus pipes:synthetic biology approaches to metabolic engineering.Metabolic engineering 2012,14(3):223-232.

4.Xu P,Li L,Zhang F,Stephanopoulos G,Koffas M:Improving fatty acids production by engineering dynamic pathway regulation and metabolic control.Proceedings of the National Academy of Sciences of the United States of America 2014,111(31):11299-11304.

5.Burda WN,Miller HK,Krute CN,Leighton SL,Carroll RK,Shaw LN:Investigating the genetic regulation of the ECF sigma factor sigma(S)in Staphylococcus aureus.Bmc Microbiol2014,14.

6.Weber W,Fussenegger M:Molecular diversity--the toolbox for synthetic gene switches and networks.Current opinion in chemical biology 2011,15(3):414-420.

7.Schujman GE,Paoletti L,Grossman AD,de Mendoza D:FapR,a bacterial transcription factor involved in global regulation of membrane lipid biosynthesis.Developmental cell2003,4(5):663-672.

8.Martinez MA,Zaballa ME,Schaeffer F,Bellinzoni M,Albanesi D,Schujman GE,Vila AJ,Alzari PM,de Mendoza D:A novel role of malonyl-ACP in lipid homeostasis.Biochemistry 2010,49(14):3161-3167.

9.Liu D,Xiao Y,Evans BS,Zhang F:Negative feedback regulation of fatty acid production based on a malonyl-CoA sensor-actuator.ACS synthetic biology 2015,4(2):132-140.

10.Merulla D,van der Meer JR:Regulatable and Modulable Background Expression Control in Prokaryotic Synthetic Circuits by Auxiliary Repressor Binding Sites.ACS synthetic biology 2016,5(1):36-45.

11.Ptashne M:Isolation of theλphage repressor.Proceedings of the National Academy of Sciences of the United States of America 1967,57(2):306.

12.Gilbert W,Müller-Hill B:Isolation of the lac repressor.Proceedings of the National Academy of Sciences of the United States of America 1966,56(6):1891.

13.Pabo CO,Lewis M:The operator-binding domain of lambda repressor:structure and DNA recognition.Nature 1982,298(5873):443-447.

14.Jain D,Nickels BE,Sun L,Hochschild A,Darst SA:Structure of a ternary transcription activation complex.Molecular cell 2004,13(1):45-53.

15.Pabo CO,Sauer RT,Sturtevant JM,Ptashne M:The lambda repressor contains two domains.Proceedings of the National Academy of Sciences 1979,76(4):1608-1612.

16.Bell CE,Frescura P,Hochschild A,Lewis M:Crystal structure of theλrepressor C-terminal domain provides a model for cooperative operator binding.Cell 2000,101(7):801-811.

17.Johnson AD,Meyer BJ,Ptashne M:Interactions between DNA-bound repressors govern regulation by theλphage repressor.Proceedings of the National Academy of Sciences1979,76(10):5061-5065.

18.Révet B,von Wilcken-Bergmann B,Bessert H,Barker A,Müller-Hill B:Four dimers ofλrepressor bound to two suitably spaced pairs ofλoperators form octamers and DNA loops over large distances.Current biology 1999,9(3):151-154.

19.Dodd IB,Perkins AJ,Tsemitsidis D,Egan JB:Octamerization ofλCI repressor is needed for effective repression of P RM and efficient switching from lysogeny.Genes&development 2001,15(22):3013-3022.

20.Hochschild A,Lewis M:The bacteriophageλCI protein finds an asymmetric solution.Current opinion in structural biology 2009,19(1):79-86.

21.Friedman AM,Fischmann TO,Steitz TA:Crystal structure of lac repressor core tetramer and its implications for DNA looping.Science 1995,268(5218):1721.

22.Cournac A,Plumbridge J:DNA Looping in Prokaryotes:Experimental and Theoretical Approaches.J Bacteriol 2013,195(6):1109-1119.

23.Becker NA,Greiner AM,Peters JP,Maher LJ:Bacterial promoter repression by DNA looping without protein-protein binding competition.Nucleic Acids Res 2014,42(9):5495-5504.

24.Hochschild A,Ptashne M:Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix.Cell 1986,44(5):681-687.

25.Jiang W,Zhao X,Gabrieli T,Lou C,Ebenstein Y,Zhu TF:Cas9-Assisted Targeting of CHromosome segments CATCH enables one-step targeted cloning of large gene clusters.Nature communications 2015,6:8101.

26.Lou C,Stanton B,Chen Y-J,Munsky B,Voigt CA:Ribozyme-based insulator parts buffer synthetic circuits from genetic context.Nature biotechnology 2012,30(11):1137-1142.

27.Stanton,B,et al.:Genomic mining of prokaryotic repressors for orthogonal logic gates.Nature Chemical Biology,2014.10(2):99-105.

Claims

1. A method of increasing the transcriptional repression ability of a repressor protein, the method comprising:

operably linking a multimerization domain to a DNA-binding domain of the repressor protein, wherein the multimerization domain is selected from the group consisting of multimerization domains of CI family repressor proteins; and

arranging the promoter that interacts with the repressor protein to comprise one or two pairs of operator sites that interact with the DNA binding domain of the repressor protein,

wherein, when the promoter comprises an operation site pair, the operation site pair is positioned at the downstream of the transcription initiation site of the promoter at the same time or positioned at two sides of the transcription initiation site of the promoter respectively; or

Wherein, when the promoter comprises two pairs of manipulation sites, a first pair of manipulation sites is located either simultaneously downstream of or on each side of the transcription initiation site of the promoter, and a second pair of manipulation sites is located either simultaneously upstream or downstream of the transcription initiation site of the promoter.

2. The method of claim 1, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

3. The method of claim 2, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

4. The method of any one of claims 1-3, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

5. The method of any one of claims 1-3, wherein the multimerization domain is directly linked to the DNA-binding domain.

6. The method of any one of claims 1-3, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

7. The method of claim 6, wherein the linker peptide is 1-20 amino acids in length.

8. The method of claim 7, wherein the linker peptide has the amino acid sequence of SEQ ID NO: 6.

9. A method of increasing the sensitivity of a gene regulatory network, the method comprising:

10. A method of improving the switching properties of a promoter in a host cell, the method comprising:

setting the promoter to comprise one or two manipulation site pairs; and

expressing in the host cell a repressor protein comprising a DNA binding domain capable of binding to the manipulation site and a multimerization domain selected from the group consisting of the multimerization domain of a CI family repressor protein,

11. The method of claim 10, wherein the promoter is the T7 promoter.

12. The method of any one of claims 9-11, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

13. The method of claim 12, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

14. The method of any one of claims 9-11, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

15. The method of any one of claims 9-11, wherein the multimerization domain is directly linked to the DNA-binding domain.

16. The method of any one of claims 9-11, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

17. The method of claim 16, wherein the linker peptide is 1-20 amino acids in length.

18. The method of claim 17, wherein the linker peptide has the amino acid sequence of SEQ ID NO: 6.

19. The method of any one of claims 9-11, wherein, when the promoter comprises one pair of manipulation sites, the two manipulation sites in the pair are spaced such that the repressor protein forms a loop upon binding to the pair of manipulation sites in tetrameric form, the pair of manipulation sites being on the same side of the loop.

20. The method of any one of claims 9-11, wherein when the promoter comprises two pairs of manipulation sites, a first pair of manipulation sites is located on each side of the transcription start site of the promoter and a second pair of manipulation sites is located both upstream and downstream of the transcription start site of the promoter.

21. The method of any one of claims 9-11, wherein the separation between the two manipulation sites in each pair of manipulation sites is 40-70 bp.

22. The method of claim 21, wherein the two manipulation sites in each pair of manipulation sites are separated by 50-70 bp.

23. The method of claim 21, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

24. The method of claim 22, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

25. The method of claim 23 or 24, wherein, when the promoter comprises two pairs of manipulation sites, the first pair of manipulation sites and the second pair of manipulation sites are separated by more than 200 bp.

26. A set of regulatory elements, comprising:

a promoter comprising one or two pairs of manipulation sites that interact with the DNA binding domain of the repressor protein,

27. The set of regulatory elements of claim 26, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

28. The set of regulatory elements of claim 27, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

29. The set of regulatory elements of any of claims 26-28, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

30. The set of regulatory elements of any of claims 26-28, wherein the multimerization domain is directly linked to the DNA-binding domain.

31. The set of regulatory elements of any of claims 26-28, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

32. The set of regulatory elements of claim 31, wherein the linker peptide is 1-20 amino acids in length.

33. The set of regulatory elements of claim 32, wherein the linker peptide has the amino acid sequence of SEQ ID NO: 6.

34. The set of regulatory elements of any one of claims 26-28, wherein, when the promoter comprises one pair of manipulation sites, the two pairs of manipulation sites are spaced such that the repressor protein forms a loop upon binding to the pair of manipulation sites in tetrameric form, the pair of manipulation sites being on the same side of the loop.

35. The set of regulatory elements of any one of claims 26 to 28, wherein when said promoter comprises one pair of manipulation sites, said pair of manipulation sites are located on each side of the transcription initiation site of said promoter, and the interval between the two of said pair of manipulation sites is 40-70 bp.

36. The set of regulatory elements according to claim 35, wherein, when said promoter comprises one pair of manipulation sites, the interval between the two manipulation sites in said pair of manipulation sites is 50-70 bp.

37. The set of regulatory elements of any of claims 26-28, wherein when said promoter comprises two pairs of manipulation sites, a first pair of manipulation sites is located on each side of the transcription initiation site of said promoter and a second pair of manipulation sites is located both upstream and downstream of the transcription initiation site of said promoter.

38. The set of regulatory elements of any of claims 26-28, wherein, when the promoter comprises two pairs of manipulation sites, the separation between the two manipulation sites in each pair of manipulation sites is 40-70 bp.

39. The set of regulatory elements according to claim 38, wherein, when the promoter comprises two pairs of manipulation sites, the separation between the two manipulation sites in each pair is 50-70 bp.

40. The set of regulatory elements of claim 38, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

41. The set of regulatory elements of claim 39, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

42. The set of regulatory elements of claim 40 or 41, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 200 bp.

43. A gene expression regulation system, the system comprising:

a second gene comprising a sequence encoding a protein to be regulated for expression of the protein to be regulated in a host cell, the second gene further comprising a promoter sequence comprising one or two pairs of manipulation sites that interact with the DNA binding domain,

44. The system of claim 43, wherein the second gene is a gene associated with a metabolic pathway.

45. The system of claim 43, wherein the CI family repressor protein is CI, CI434, HK022CI, TP901CI, or P22C 2.

46. The system of claim 45, wherein the amino acid sequence of the multimerization domain of the CI family repressor protein is SEQ ID NO: 1-SEQ ID NO: 5.

47. The system of any one of claims 43-46, wherein the multimerization domain is operably linked to the C-terminus of the DNA-binding domain.

48. The system of any one of claims 43-46, wherein the multimerization domain is directly linked to the DNA-binding domain.

49. The system of any one of claims 43-46, wherein the multimerization domain is linked to the DNA-binding domain using a linking peptide.

50. The system of claim 49, wherein the linker peptide is 1-20 amino acids in length.

51. The system of claim 50, wherein the linker peptide has the amino acid sequence of SEQ ID NO: 6.

52. The system of any one of claims 43-46, wherein, when the promoter comprises one pair of manipulation sites, the separation between the two manipulation sites in the pair of manipulation sites is such that the repressor protein forms a loop upon binding to the pair of manipulation sites in tetrameric form, the pair of manipulation sites being on the same side of the loop.

53. The system of any one of claims 43-46, wherein, when said promoter comprises one pair of manipulation sites, said pair of manipulation sites are located on each side of the transcription start site of said promoter, and the separation between the two of said pair of manipulation sites is 40-70 bp.

54. The system of claim 53, wherein, when the promoter comprises one pair of manipulation sites, the separation between the two manipulation sites in the pair of manipulation sites is 50-70 bp.

55. The system of any one of claims 43-46, wherein, when said promoter comprises two pairs of manipulation sites, a first pair of manipulation sites is located on each side of the transcription start site of said promoter and a second pair of manipulation sites is located both upstream and downstream of the transcription start site of said promoter.

56. The system of any one of claims 43-46, wherein, when the promoter comprises two pairs of manipulation sites, the separation between the two manipulation sites in each pair of manipulation sites is 40-70 bp.

57. The system of claim 56, wherein, when the promoter comprises two pairs of manipulation sites, the separation between the two manipulation sites in each pair is 50-70 bp.

58. The system of claim 56, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

59. The system of claim 57, wherein, when said promoter comprises two pairs of manipulation sites, the separation between said first pair of manipulation sites and said second pair of manipulation sites is greater than 150 bp.

60. The system of claim 58 or 59, wherein, when the promoter comprises two pairs of manipulation sites, the first pair of manipulation sites and the second pair of manipulation sites are separated by more than 200 bp.