Construction method for modular orthogonal high-performance transcription factor DNA-binding domain system

Abstract

Provided is a construction method for a modular orthogonal high-performance transcription factor DNA-binding domain (DBD) system, comprising the following steps: S1, selecting a basic architecture of a modular transcription factor and a basic architecture of a corresponding regulatory promoter; S2, on the basis of the basic architecture of the transcription factor and the basic architecture of the regulatory promoter, constructing a standard test vector containing a lethal gene CCDB, and constructing paired DBD-operator test plasmids; and S3, constructing a standard test system, testing regulatory fold-changes of different strain transcription factor DBD-operators, selecting transcription factor DBD-operators capable of high-performance transcription to perform an orthogonality test, and obtaining transcription factor DBD-operators which are mutually orthogonal and capable of high-performance transcription regulation. According to the construction method, modular DBD-operators are tested and screened to finally obtain DBD-operators which are mutually orthogonal and capable of high-performance transcription regulation.

Description

A modular orthogonal method for constructing a highly efficient transcription factor DNA binding domain system Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for constructing a modular orthogonal high-efficiency transcription factor DNA binding domain system. Background Art

[0002] During gene transcription, RNA polymerase recognizes specific DNA sequences within the promoter to initiate gene transcription. Transcription factors can bind to specific sites on DNA, affecting the probability of RNA polymerase binding to the promoter, thereby influencing the transcription rate and regulating the transcription of different genes (Alon, U., An introduction to systems biology: design principles of biological circuits. 2006: Chapman and Hall / CRC.). The different behaviors of transcription factors can be carried out by different structural and functional modular domains. Among them, the DNA binding domain (DBD), which recognizes specific DNA sequences, plays a leading role in this process. The behavior of transcription factors is described by the Hill equation, and the regulation of transcription factors can be optimized by optimizing its parameters. The greater the affinity of the transcription factor for binding to the promoter and the larger the Hill coefficient n, the stronger the transcription factor's regulation and the better its regulatory performance (Marchisio and M. Andrea, [Learning Materials in Biosciences] Introduction in Synthetic Biology. 2018.).

[0003] In addition to the above-mentioned parameter optimization, increasing the synergy of transcriptional regulation by increasing the regulatable dimerization process before transcription factors bind to the promoter can also play a role in optimizing regulatory performance. This idea comes from the natural structure of LexA protein: LexA protein is the main regulatory factor of bacterial SOS response, composed of 202 amino acids, its amino-terminal domain and carboxyl-terminal domain are separated and connected by a relatively flexible linker sequence. Its carboxyl-terminal domain contains dimerization activity and self-cleavage activity, and can self-cleave at position 84-85 (Luo, Y., et al., Crystal Structure of LexA: A Conformational Switch for Regulation of Self-Cleavage. Cell, 2001.106(5): p.585-594.); the amino-terminal domain contains a winged-HTH motif, which is responsible for binding to DNA sequences. When LexA exists in the form of a dimer, the winged-HTH of each monomer binds to one half site and stably binds to the SOS box (Zhang, AP, YZ Pigli, and PARice, Structure of the LexA-DNA complex and implications for SOS box The consensus sequence of the E. coli SOS box consists of 16 bases: CTGT(N)8ACAG, which contains two inverted palindromic half-sites separated by an 8-base spacer sequence, which serve as key bases for recognition (Walker, GC, Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev, 1984. 48(1): p. 60-93.). Compared with the dimeric form, the binding ability of monomeric LexA is greatly reduced (Kim, B. and JW Little, Dimerization of a specific DNA-binding protein on the DNA. Science, 1992. 255(5041): p. 203-6.). Therefore, when the LexA protein self-cleaves, the amino-terminal domains of the two monomers cannot dimerize and thus dissociate from the SOS box.Therefore, the amino terminus of LexA can be used as a functionally independent modular DNA-binding domain, fused with different carboxyl-terminal dimerization domains to perform site-specific DNA recognition and binding in the dimerized state. By using dimerization domains induced by different conditions, gene expression regulation in response to different signals can be achieved.

[0004] Artificially synthesized transcription factors often use zinc fingers, TALEs, and CRISPR-dCas9 as custom DNA binding modules, connecting functional domains such as ligand binding domains or transcription activation domains for construction (Khalil, Ahmad S., et al., A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions. Cell, 2012. 150(3): p. 647-658.). Based on three zinc finger structures binding to 9bp DNA, Khalil et al. designed a series of orthogonal synthetic transcription factor-operator sequence pairs, and carried out a series of subsequent optimization processes to construct a eukaryotic transcription regulation system with zinc fingers as the core (Bashor, CJ, et al., Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies. Science, 2019. 364(6440): p. 593-597.). By using a high-affinity zinc finger DNA binding module, the performance of the transcription factor itself can be optimized, and without changing the characteristics of the transcription factor itself, the output of transcriptional regulation can be optimized by increasing the Hill coefficient. In addition, the cooperativity in the transcriptional regulation process can be increased by increasing the interaction between a single zinc finger transcription activator and a zinc finger DNA binding protein. Through the controllable interaction between the two proteins, the parameters of transcription factor binding to DNA can be changed to obtain a transcription factor complex with higher affinity, thereby optimizing the transcriptional regulation output (Bashor, CJ, et al., Complex signal processing in synthetic gene circuits using cooperative regulatory assemblies. Science, 2019. 364(6440): p.593-597.).

[0005] In addition, the prokaryotic SOS response regulator LexA protein is also commonly used as a DNA binding domain for the construction of artificial transcription factors. The yeast artificial transcription factor XEV was originally modified from GAL4 by replacing the GAL4 LBD domain with the ER LBD and VP16, and was named GEV. GEV retains the DNA binding domain of natural GAL4 and has regulatory crosstalk with natural GAL4 (Boch, J., et al., Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 2009. 326(5959): p.1509-12.). Subsequently, McIsaac, RS, et al. further replaced the DNA binding domain of GAL4 with a modular zinc finger domain, thereby reducing growth toxicity, but the regulatory effect was poor, with a fold change of no more than 10 times (Deng, D., et al., Structural basis for sequence-specific recognition of DNA by TAL effectors. Science, 2012. 335(6069): p.720-3.). Ottoz, DS, et al. further replaced the zinc finger structure with the full-length LexA protein as the DNA binding domain, and replaced different transcription activation domains, achieving better regulatory effects (Gaj, T., CAGersbach, and CF Barbas, 3rd, ZFN, TALEN, and CRISPR / Cas-based methods for genome engineering. Trends Biotechnol, 2013. 31(7): p.397-405.).

[0006] A common method for obtaining orthogonal transcription factors is to establish a mutation library and screen for functional orthogonal transcription factor pairs. Andreas K. et al. used cI to regulate the expression of key genes of Mu13 phage and screened the cI functional library, from which 12 mutually orthogonal mutations were screened, which can be applied to the dual activation-repression system ( AK, A. Jaramillo, and M. Isalan, Engineering orthogonal dual transcription factors for multi-input synthetic promoters. Nature Communications, 2016. 7(1): p. 13858.). In addition, Stanton, BC et al. screened 73 transcription factors in the TetR family in 2014 and identified 16 orthogonal transcription factor-promoter pairs for constructing logic gate gene circuits (Stanton, BC, et al., Genomic mining of prokaryotic repressors for orthogonal logic gates. Nat Chem Biol, 2014. 10(2): p. 99-105.). In 2018, Nomura, Wataruden, et al. used DNA binding domains TALE and CRISPR-dCas9 with custom recognition sites, and fused them with rapamycin and gibberellin induction systems, respectively, to achieve orthogonal control of gene expression (Nomura, W., et al., Efficient and Orthogonal Transcription Regulation by Chemically Inducible Artificial Transcription Factors. Biochemistry, 2018. 57(45): p. 6452-6459.).

[0007] Complex gene circuits require multiple pairs of transcription factors with excellent regulatory properties, mutually orthogonal, and non-crosstalking functions. However, current transcriptional regulatory systems in eukaryotic cells still primarily utilize endogenous constitutive promoters to regulate expression. Transcriptional regulatory tools controlled by transcription factors are primarily derived from prokaryotic systems, such as LacI, TetR, and XylR, or are synthetic modular transcription factors. These tools are limited in number, resulting in high background leakage, limited activation, and potential crosstalk with endogenous pathways. In commonly used transcriptional expression systems, transcription factors recognize and bind regulatory sequences as integrated monomers or multimers. These factors are often optimized with multiple binding sites, resulting in limitations in terms of fold change. Commonly used modular DNA-binding domains, including TALEs, ZFs, and CRISPR / dCas9, require extensive effort to construct orthogonal DBD libraries, resulting in a limited number of efficient tools. Furthermore, the simultaneous use of multiple transcriptional regulatory systems requires orthogonality between transcription factors. Transcription factors act as converters between extracellular and intracellular biological signals, and their DNA-binding domains serve as crucial interfaces in this process. Therefore, finding multiple pairs of mutually orthogonal high-performance DNA binding domains and their recognition sequence pairs is very important for constructing complex gene circuits.

[0008] Summary of the Invention

[0009] The present invention provides a method for constructing a modular orthogonal high-efficiency transcription factor DNA binding domain system, which solves the problems of poor transcription factor regulation effect, limited types and crosstalk in metabolic engineering and cell circuit design in the prior art.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] A method for constructing a modular orthogonal high-efficiency transcription factor DNA binding domain system comprises the following steps:

[0012] S1. Select the basic architecture of modular transcription factors and the basic architecture of their regulated promoters;

[0013] S2. Based on the basic structure of transcription factors and regulatory promoters, a standard test vector containing the lethal gene CCDB is constructed. The externally obtained DNA binding domain (DBD) and operator sequences are inserted into the lethal gene CCDB portion of the standard test vector to construct a paired DBD-operator test plasmid;

[0014] S3. Integrate the constructed DBD-operator test plasmid and reporter gene plasmid into the strain to obtain a test strain; culture the test strain to detect the expression intensity of the reporter gene, test the regulation fold of the transcription factor DBD-operator in different strains, select the transcription factor DBD-operator with efficient transcription, and conduct orthogonality testing to obtain the transcription factor DBD-operator with mutual orthogonality and efficient transcription regulation.

[0015] In the present invention, the basic structure of the modular transcription factor is the E. coli transcription repressor and the Saccharomyces cerevisiae transcription activator; the structure of the E. coli transcription repressor is the lexAec in E. coli. 1-87 -RpaR 1-179 , whose sequence is shown in SEQ ID NO: 1; the structure of the Saccharomyces cerevisiae transcription activator is lexAec 1-87 -RpaR 1-179 - VP16, the sequence of which is shown in SEQ ID NO: 2.

[0016] Furthermore, the promoter of the E. coli transcription repressor is a promoter in which operators corresponding to different DBDs are placed at the +1 position of the promoter as its regulated downstream promoter; the promoter of the Saccharomyces cerevisiae transcription activator is a promoter in which the Saccharomyces cerevisiae promoter proADH2 is used as the basic skeleton, and its regulatory sequence is replaced with operators corresponding to different DBDs as its regulated downstream promoter.

[0017] In the present invention, based on the basic structure, S2 constructs a standard test vector containing the lethal gene CCDB by replacing the DBD part of the transcription factor with the lethal gene CCDB, and constructs standard test vectors for E. coli and Saccharomyces cerevisiae, respectively.

[0018] Furthermore, the DBD portion of the transcription factor is controlled by pllac (IPTG induction) and T7 terminator (T7t) in the E. coli system to express the transcription factor; and the DBD portion of the transcription factor is controlled by ptet (aTc induction) and tENO2 terminator in the Saccharomyces cerevisiae system to express the transcription factor.

[0019] Furthermore, the standard test vector sequence of E. coli is constructed as SEQ ID NO: 3, and the standard test vector sequence of Saccharomyces cerevisiae is constructed as SEQ ID NO: 4.

[0020] In the present invention, the DNA binding domain and operator sequence are obtained by mining protein and operator sequence information in a database.

[0021] In the present invention, the reporter gene plasmid uses yellow fluorescent protein YFP as the reporter gene in both E. coli and Saccharomyces cerevisiae systems.

[0022] In the present invention, the strains are Saccharomyces cerevisiae strains and E. coli strains.

[0023] Furthermore, the E. coli strain test culture process is as follows: the E. coli strain transformant to be tested is inoculated in the LB medium with the corresponding resistance, and then transferred to the M9 Gly medium containing the corresponding concentration of the corresponding inducer and the corresponding resistance for the first time, and cultured until the OD 600 The first E. coli plate reader test was performed using a plate reader when the OD600 was ≈0.3 (about 5 hours). At the same time, the cells were transferred to M9 Gly medium containing the corresponding concentration of inducer and corresponding resistance, cultured to OD600 ≈0.3 (about 5 hours), and the second E. coli plate reader test was performed. At the same time, a blank control MG1655 ALT and a constitutively expressed positive control MG1655 ALT / BZC041 were set up, and three replicate sample wells were set up for each treatment.

[0024] Furthermore, the test culture process of the brewer's yeast strain is as follows: the yeast strain to be tested is inoculated into a medium containing SD-ΔLEU-ΔURA for culture, and then transferred to SD medium, and the corresponding inducer at the corresponding concentration is added. After induction culture, samples are taken, and the test bacterial liquid is diluted with PBS buffer and tested using a flow cytometer; each experiment sets up a blank negative control CYE72, a constitutive expression positive control CYE72 / CY671 (as an expression standard control), and an inducible expression positive control CYE72 / CY637 (aTc-induced expression, used to calibrate the inducer concentration), among which CYE72 / CY637 should be added with 100 ng / ml aTc during transfer to induce expression of the reporter gene, and three replicate sample wells are set for each treatment.

[0025] A modular orthogonal high-efficiency transcription factor DNA binding domain system is obtained through the above construction method.

[0026] The present invention has the following beneficial effects:

[0027] (1) The present invention provides a method for constructing a modular orthogonal and efficient transcription factor DNA binding domain system. The basic architecture of the modular transcription factor is determined, and then a labeled test vector and a test system are constructed to test and screen the modular DBD-operators, ultimately obtaining mutually orthogonal and highly efficient transcriptional regulatory DBD-operators.

[0028] (2) The present invention determines the feasibility of modularization of transcription factor DBDs and the high efficiency of cooperative regulation of transcription factor dimerization; a series of highly efficient DBD-operator pairs from different host sources and different protein families, which are mutually orthogonal, are obtained to solve the problems of few transcriptional regulatory tools and limited regulatory multiples, and also avoid the mutual crosstalk between the host endogenous and transcription factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods.

[0030] Figure 1 is a schematic diagram of the modular transcription factor architecture of E. coli and Saccharomyces cerevisiae.

[0031] Figure 2 is a schematic diagram of the DBD screening transcription factors and corresponding promoter architectures for E. coli and Saccharomyces cerevisiae.

[0032] Figure 3 shows the standard test vector for DBD screening in E. coli and Saccharomyces cerevisiae.

[0033] Figure 4 shows the database mining workflow and results.

[0034] Figure 5 shows lexAec 1-87 Schematic diagram of the DBD domain structure.

[0035] Figure 6 shows the standard test system for DBD screening in E. coli and Saccharomyces cerevisiae.

[0036] Figure 7 shows the preliminary screening test results of lexA strains.

[0037] Figure 8 shows the preliminary orthogonality test results of 1exA strains.

[0038] Figure 9 shows the DBD screening test and classification.

[0039] FIG10 shows the induction folds of E. coli DBD screening results.

[0040] FIG11 shows the results of the DBD screening test of Saccharomyces cerevisiae.

[0041] FIG12 shows the orthogonality test results between different DBDs. DETAILED DESCRIPTION

[0042] The present invention is based on the cooperative regulation of transcription factor dimers, and provides a set of mutually orthogonal modular efficient DNA binding domains as a tool library, which can effectively solve the problems of poor transcription factor regulation effect, few types, and crosstalk in metabolic engineering and cell circuit design. At the same time, the DNA binding domain in the tool library can connect the protein domains of different protein interactions, as an interface between protein interaction and gene expression output, to provide a variety of effective operations for gene expression regulation. The present invention takes the inducer response dimerization domain (1-179aa) of the group response transcription regulatory protein RpaR in Rhodopseudomonas palustris as an example, and screens the regulatory effects of different DNA binding domain-operator pairs.

[0043] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0044] The Golden Gate system and conditions used in the following examples are as follows:

[0045] The E. coli test culture conditions in the following examples are as follows:

[0046] After obtaining E. coli transformants, they were inoculated into 200 μl of LB medium with the corresponding resistance in a 96-well U-shaped plate and cultured at 37°C and 800 rpm overnight. They were first transferred to M9 Gly medium containing the corresponding inducer and the corresponding resistance at a ratio of 1:200. At the same time, each experimental group was set up as a non-induced group, an inducer IPTG+- (working concentration: 100 μM) or p-coumaroyl homoserine lactone pC-HSL-+ (working concentration: 20 μM), or both groups were added. The culture was carried out at 37°C and 800 rpm until the OD 600 The first preliminary screening test was performed using a plate reader at ≈0.3 (about 5 hours). For subsequent effective DBDs, the second transfer was continued at a ratio of 1:20 to M9 Gly medium containing the corresponding concentration of inducer and corresponding resistance, and cultured at 37°C and 800 rpm until OD 600 ≈0.3 (about 5h), conduct the second test. OD with 200 PRO platereader 600 The fluorescence value of each well was calculated as follows: YFP = test 测试 / OD 600测试 -testMG1655 / OD 600MG1655 The relative fluorescence expression of each experimental group was calculated as RPU = YFP 测试 / YFP MG1655ALT / BZC041 The calculation method for the inhibition fold of transcription factors is: Fold Change = RPU -- / RPU ++ .

[0047] The yeast test culture conditions in the following examples are as follows:

[0048] After obtaining the yeast transformants, they were inoculated into a 96-deep-well plate containing 500 μl of the corresponding defective SD medium and cultured at 30°C and 800 rpm for 24-48 hours. Then, they were transferred to a new 96-deep-well plate at a ratio of 1:200. At the same time, each experimental group was set up as a non-induced group, an inducer anhydrotetracycline aTc+- (working concentration: 100 ng / ml) or p-coumaryl homoserine lactone pC-HSL-+ (working concentration: 100 μM), or both were added ++ group. After culturing for 16 hours, an appropriate amount of bacterial solution was taken and diluted with 1xPBS solution. The results were analyzed using a flow cytometer BD FACSCelesta TM The FITC-A channel was used to detect the expression of the fluorescent protein YFP in 10,000 cells. The data were processed using FlowJo, and the median was taken as the fluorescence value of each sample well. Using CYE72 / CY671 as the expression control, the relative fluorescence expression of each experimental group was calculated as follows: RPU = (YFP 测试 -YFP CYE72 ) / (YFP CYE72 / CY637 -YFP CYE72 The calculation method of the induction fold of the transcription factor is: Fold Change = RPU ++ / RPU -- .

[0049] Example 1: Determining the basic structure of transcription factors

[0050] For the E. coli transcription repressor, literature research and preliminary tests confirmed the use of the dimerization domain 1-179aa of the quorum sensing transcription factor RpaR of Rhodopseudomonas palustris. This domain was connected to the 1-87aa domain of the E. coli ilexA protein through a flexible linker (GGGGS)3 to form a domain-modular artificial transcription factor lexAec. 1-87 -RpaR 1-179 (SEQ ID NO: 1). This transcription factor can regulate the promoter containing lexA operatorlexO1, and the promoter sequence is: The bold characters are -35 and -10 regions, and the uppercase characters are the lexO1 sequences. 1-179 Able to form dimers in response to pC-HSL. 1-87 -RpaR 1-179 Expression of the reporter gene is inhibited only when a certain concentration of pC-HSL is added. It is important to note that, in this promoter sequence, the operator is placed between the -35 and -10 positions of the promoter, as well as at the +1 position. In this method, subsequent screening is performed to select one of these positions for operator placement. This establishes the basic architecture of transcription factors and downstream regulatory promoters in the E. coli system.

[0051] For the Saccharomyces cerevisiae transcriptional activator, based on the existing transcription factor and reporter gene circuit construction in the laboratory, similar to E. coli, the transcription factor lexAec was confirmed 1-87 -RpaR 1-179 -VP16 (SEQ ID NO: 2) and the corresponding regulatory promoter: The non-bold, capitalized portion is the operator, while the bold portion represents the polyA, TATA box, TSS, and Kozak sequence that form the basic promoter structure. Note that this promoter contains two operators, a format also used for the yeast promoter in this method, allowing for screening by placing different operators at the operator position.

[0052] Example 2: Construction of standard test vector

[0053] 1. Construction of E. coli transcriptional repressor DBD screening vector LXR78

[0054] Using the laboratory-available plasmid CY386 as a template, primers CCDB-78-F (5'-TTCTAATGTGAGACCTTATATTCCCCAGAACATCAGGTTAATGG-3') and CCDB-78-R (5'-CGCCACCAGAGACCGGCTTACTAAAAGCCAGATAACAGTATGC-3') were used to amplify the CCDB lethal gene. PCR fragment 2 was obtained by amplifying the laboratory-available plasmid HC105 with primers RpaR179-78-F (5'-CAGTGAGGTCTCTGGTGGCGGCGGTAGCGGTGGAGGGGGGTCTGGTGGAGGAGGCTCAATTGTGGGTGAAGATCAGCTGTG-3') and RpaR179-78-R (5'-TAAGCCGGTCTCATTTAATTACGACGAATCGGTTTCGGACG-3'). The existing plasmid BZC008 in the laboratory was used as the vector, fragments 1 and 2 were used as inserts, digested with BsaI at 37°C for 30 min, inactivated at 80°C for 20 min, and ligated with T4 ligase at 16°C for 2 h to obtain the E. coli transcription repressor DBD screening vector LXR78, see SEQ ID NO: 4.

[0055] 2. Construction of the Saccharomyces cerevisiae transcriptional activator DBD screening vector LXR66

[0056] Using the lexAec 1-87 -RpaR 1-179 -VP16 expression plasmid pXJH119 was used as a template and primers 66-F (5'-GGTTCTAAGGATATCTCTGCTGGAGACATGA-3') and 66-R (5'-CATTTTTTATTTATTTTTGTAGCTTGATATTCTCTATCAC-3') were used to amplify PCR fragment 1. Using the laboratory-available plasmid CY386 as a template, primers Homo-CCDB-66-F (5'-ACAAAAATAAATAAAAAATGAGGTCTTCTTATATTCCCCAGAACATCAGGTTAATGG-3') and Homo-CCDB-66-R (5'-GCAGAGATATCCTTAGAACCAGGTCTTCGGCTTACTAAAAGCCAGATAACAGTATGC-3') were used to amplify the CCDB lethal gene. The two PCR fragments were homologously recombined using II One Step Cloning Kit C112 to obtain the Saccharomyces cerevisiae transcription activator DBD screening vector LXR66, as shown in SEQ ID NO: 3.

[0057] Example 3: DBD-operator mining

[0058] LexA operon sequences from various bacterial species, obtained by crawling the website https: / / regprecise.lbl.gov / search.jsp, were classified by bacterial phylum. The PWMs of all LexA operon sequences from different phyla were calculated and consensus sequences were obtained. Since sequence lengths vary among bacterial species, a sequence alignment algorithm was first used to align the sequences, followed by PWM calculation and logo generation. The DBD mining process and a partial logo image are shown in Figure 4. Based on the PWMs obtained from the classifications, potentially consistent sequences were listed. Once the consensus sequences were obtained, these consensus sequences were entered into the MEME and collecTF databases for sequence alignment, resulting in the corresponding LexA TFs for experimental verification.

[0059] Example 4: Preliminary testing of lexA family transcription factors

[0060] 1. DBD domain extraction

[0061] Based on the corresponding protein IDs, protein gene sequences and structural information were retrieved from the Uniprot (https: / / www.uniprot.org / ) database. Protein structures were observed and extracted by amino acid sequence alignment and pymol software. After obtaining the gene sequences corresponding to the extracted protein domains, the BpiI and BsaI restriction sites were eliminated through synonymous mutations. After obtaining the DBD gene sequences with the restriction sites eliminated, the sequences 5'-cagtgaggtctctaatgtct-3' and 5'-ggtgtgagaccggctta-3' were added to the 5' end and the 3' end, respectively, to add BsaI restriction site recognition sites and cohesive end sequences corresponding to the vector LXR78. The resulting DBD sequences, endowed with BsaI restriction sites, were synthesized at Qingke Biotechnology. The resulting proteins were named lexA + species name + extracted amino acids. The DBD amino acid sequences are shown in SEQ ID NOs: 5-15.

[0062] 2. Construction of E. coli test system with different DBD-operators

[0063] Using the synthesized lexA strain DBD sequence as an insert and LXR78 as a vector, the CCDB lethal gene sequence was replaced with a different DBD gene sequence using the BsaI Golden Gate technique to generate Golden Gate reaction products. These constructs were transformed into the laboratory strain DH10B 12S to obtain clones. Following conventional plasmid extraction and sequencing, plasmids expressing transcription factors for lexA strain DBD screening were obtained and named LXR33-LXR43, respectively.

[0064] For reporter gene plasmids, the promoter contains the operatorlexO strains corresponding to the DBD binding of lexA strains. The operator sequence and corresponding protein and host information are shown in Table 1:

[0065] Table 1 Operator sequence and corresponding protein and host information

[0066] A promoter sequence with BsaI residues at both ends was obtained by long-primer PCR. Using the existing plasmid CWJB002 in the laboratory as a vector, a series of reporter gene plasmids were generated using BsaI Golden Gate technology. The PCR primer information for promoter synthesis is shown in Table 2:

[0067] Table 2 PCR primer information for promoter synthesis

[0068] 3. Testing the regulation fold of different E. coli transcription factor DBD-operators

[0069] The 12 DBD-operator screening plasmids obtained were co-transformed into the existing host strain MG1655 ALT in the laboratory through chemical transformation to obtain E. coli test transformants. Initial screening and test culture in E. coli yielded a test bacterial solution, which was then tested using a plate reader. The test data are shown in Figure 7. Figure 7 shows that all 12 pairs of lexA family DBD-operators exhibited varying degrees of good regulation.

[0070] It is important to note that in preliminary testing, lexO strains were inserted at one of two positions: ① between the -35 and -10 regions, or ② at the +1 position. During construction, differences in operator sequences between the -35 and -10 regions can significantly alter promoter strength, leading to growth stress caused by YFP expression and thus construction difficulties. Given the toxicity associated with the promoter's background expression strength, most subsequent E. coli constructs adopted a promoter structure that placed the operator at the +1 position of the core promoter.

[0071] 4. Orthogonality test

[0072] The 12 DBD screening transcription factor expression plasmids and 12 corresponding reporter gene plasmids were partially aligned and transformed into MG1655 ALT, yielding 37 E. coli test transformants for preliminary testing of interactions and orthogonality between different DBD × operator combinations. Initial E. coli screening cultures were used to generate test cultures, which were then analyzed using a plate reader. The test data are shown in Figure 8. Figure 8 shows that the DBD-operator combinations can be divided into three mutually orthogonal groups, with good orthogonality between the three groups, while none of the groups are orthogonal within each other. Observation also reveals that the 12 operator sequences can be divided into three corresponding categories based on half-site sequence, orientation, and the length of the spacer between the two half sites: two opposing CTGTs spaced 8 bp apart (exemplified by E. coli), two opposing GAACs spaced 4 bp apart (exemplified by B. subtilis), and two GTTCs spaced 6 bp apart in the same orientation (exemplified by S. meliloti). Variation in any of these factors can produce orthogonal interactions. This pattern provides guidance for the subsequent selection of orthogonal DBDs and operators.

[0073] Example 5: Screening and testing of transcription factor DBD-operators from different hosts and protein families

[0074] 1. Construction of E. coli test system with different DBD-operators

[0075] Based on the results of the preliminary screening test, transcription factors from different prokaryotic protein families from the lexA family database mining, phage, and GroovDB database (https: / / www.groov.bio / ) were further selected (see Table 3).

[0076] Table 3 Transcription factors of different prokaryotic protein families

[0077] Following the same method as above, potential DNA-binding domains (amino acid sequences shown in SEQ ID NOs: 16-44) were extracted and qualitatively tested for interaction with the corresponding operators. DNA fragments containing BsaI restriction enzyme recognition sites and cohesive end sequences corresponding to the LXR78 vector were obtained via gene synthesis, pre-existing sequence PCR, or colony PCR. Using LXR78 as the vector, the CCDB lethal gene sequence was replaced with different DBD gene sequences using the BsaI Golden Gate technique to generate Golden Gate reaction products. The constructed products were transformed into the laboratory strain DH10B 12S to obtain clones. Following conventional plasmid extraction and sequencing, plasmids expressing transcription factors for different DBD screening were obtained. Similarly, promoter fragments were obtained via long-primer PCR, and a series of reporter gene plasmids were generated using the BsaI Golden Gate technique using CWJB002 as the vector. The forward primers used for promoter synthesis (not indicated) were all the same lexOs+1-F as described above. PCR reverse primers and some forward primer information are shown in Table 4:

[0078] Table 4 PCR reverse primer and some forward primer information

[0079] 2. Testing the regulation fold of different E. coli transcription factor DBD-operators

[0080] The 12 DBD-operator screening plasmid pairs obtained were chemically transformed into the existing host strain MG1655 ALT in the laboratory to obtain E. coli test transformants. Initial screening and test culture in E. coli yielded a test culture. Plate reader testing yielded test data, as shown in Figure 9. Fifteen orthogonal DBDs with promising regulatory effects were further tested for 10 hours of induction, with induction fold data shown in Figure 10.

[0081] 3. Construction of an Effective DBD-operator Test System in Saccharomyces cerevisiae

[0082] Fifteen pairs of DBD operators with promising regulatory effects, screened in E. coli, were placed in yeast for the construction of a standard screening system. To construct transcription factor expression plasmids, the corresponding E. coli expression plasmids were used as templates. PCR amplification was performed to obtain a DBD fragment with the corresponding BpiI restriction site for the LXR66 vector and cohesive ends: 5'-cagtgaGAAGACCTaatgtct-3' at the 5' end and 5'-ggttAGGTCTTCggctta-3' at the 3' end. Using LXR66 as a vector, the CCDB lethal gene sequence was replaced with a different DBD gene sequence using the BpiI Golden Gate technique. See Table 5 for DBD plasmid information.

[0083] Table 5 Different DBD plasmid information

[0084] For the reporter gene plasmid, we used the existing lab-derived plasmid pXJH1 as a construct, replacing the corresponding operators with the regulatory regions corresponding to the yeast promoters described above. After synthesizing two fragments, the upstream regulatory sequence and the downstream core promoter sequence, by PCR, we then used overlap PCR to obtain a complete yeast promoter fragment with the corresponding BpiI restriction sites and sticky ends at both ends. Using pXJH1 as a vector, we replaced the CCDB lethal gene sequence with different yeast promoter sequences using the BpiI Golden Gate technique. Promoter sequence information is shown in Table 6:

[0085] Table 6 Sequence information of different yeast promoters

[0086] After obtaining 15 pairs of screening plasmids, they were digested with restriction endonuclease BsaI to obtain the transformed enzyme fragments. TM The yeast transformation kit was used to prepare the CYE72 competent strain, and the enzyme-digested fragments were then transferred into the CYE72 competent strain in two steps to obtain yeast transformants.

[0087] 4. Testing the Regulation Fold of the Saccharomyces cerevisiae Transcription Factor DBD-operator

[0088] The test bacterial solution was obtained by yeast test culture and BD FACSCelesta TMFlow cytometry analysis yielded test data shown in Figure 11. As shown in Figure 11, most DBDs effective in E. coli also exhibit transcriptional regulatory functions in Saccharomyces cerevisiae. However, the expression of different DBDs in the two hosts is inconsistent and orderly, likely due to differences in the host internal environments. This resulted in the identification of 12 DBD-operator pairs with high transcriptional regulatory potential in Saccharomyces cerevisiae for downstream applications and testing.

[0089] Example 6: Orthogonality test of valid DBD and operator

[0090] After screening in E. coli and Saccharomyces cerevisiae, 12 DBD-operator pairs with high transcription efficiency were identified and tested for orthogonality. The corresponding 12 transcription factor expression plasmids and 12 reporter gene plasmids were fully arrayed and double-transformed into MG1655 ALT cells, yielding 144 transformants for DBD × operator orthogonality testing. Test cultures were obtained through E. coli screening and plate reader analysis, yielding an orthogonal table (Figure 12). Each group represents the fold-regulation under the corresponding DBD × operator conditions, with darker colors indicating greater fold-regulation. In the case of orthogonality, the orthogonal table should contain values ​​only on the diagonal. As shown in the figure, all 12 DBD-operator pairs exhibit good orthogonality, consistent with the information obtained from the operator sequences. This identification of 12 mutually orthogonal, highly efficient DBD-operator pairs in Saccharomyces cerevisiae provides a foundation for downstream testing and the application of multi-transcription factor regulatory systems.

[0091] This method introduces synergistic regulation by linking different DBDs to an inducible dimerization domain. The system then screens the different DBDs for transcriptional regulation, performing orthogonality testing and extracting orthogonal features to generate multiple high-affinity DBD-operator pairs. In this system, each DBD monomer binds to a regulatable dimerization domain and, under appropriate induction conditions, dimerizes to form a dimer containing two DBDs. These dimers then recognize and bind to promoters containing an operator with two halfsite recognition sites, leading to efficient transcriptional regulation.

[0092] sequence:

[0093] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for constructing a modular orthogonal and highly efficient transcription factor DNA binding domain system, characterized in that It includes the following steps: S1. Select the basic architecture of the modular transcription factor and the basic architecture of its regulatory promoter; S2. On the basis of the basic architectures of the transcription factor and the regulatory promoter, construct a standard test vector containing the lethal gene CCDB, insert the externally obtained DNA binding domain and operator sequence into the CCDB part of the standard test vector, and construct paired DBD-operator test plasmids; S3. Co-integrate the constructed DBD-operator test plasmid and the reporter gene plasmid into the strain to obtain a test strain; Carry out test culture on the test strain, detect the expression intensity of the reporter gene, test the regulation multiples of different strain transcription factor DBD-operators, select the transcription factor DBD-operators with high-efficiency transcription for orthogonality tests, and obtain mutually orthogonal and high-efficiency transcription-regulating transcription factor DBD-operators.

2. The construction method of the modular orthogonal high-efficiency transcription factor DNA binding domain system according to claim 1, characterized in that, The basic architecture of the modular transcription factor is the E. coli transcription repressor and the Saccharomyces cerevisiae transcription activator; The E. coli transcription repressor structure is lexAec in E. coli 1-87 -RpaR 1-179 , and its sequence is as shown in SEQ ID NO: 1; the Saccharomyces cerevisiae transcription activator structure is lexAec 1-87 -RpaR 1-179 -VP16, and its sequence is as shown in SEQ ID NO:

2.

3. The construction method of the modular orthogonal and highly efficient transcription factor DNA binding domain system according to claim 2, characterized in that, The promoter of the E. coli transcription repressor is to place the operator corresponding to different DBDs at the +1 position of the promoter as its downstream regulatory promoter; the promoter of the Saccharomyces cerevisiae transcription activator is to use the Saccharomyces cerevisiae promoter proADH2 as the basic backbone and replace its regulatory sequence with the operator corresponding to different DBDs as its downstream regulatory promoter.

4. The construction method of the modular orthogonal and efficient transcription factor DNA binding domain system according to claim 2, wherein In S2, on the basis of the basic architecture of the transcription factor, the construction of the standard test vector containing the lethal gene CCDB is to replace the DBD part of the transcription factor with the lethal gene CCDB respectively, and construct the standard test vectors of E. coli and Saccharomyces cerevisiae respectively.

5. The construction method of the modular orthogonal and highly efficient transcription factor DNA binding domain system according to claim 4, characterized in that, For the DBD part of the transcription factor, in the E. coli system, the expression of the transcription factor is controlled by pllac and the T7 terminator; for the DBD part of the transcription factor, in the Saccharomyces cerevisiae system, the expression of the transcription factor is controlled by ptet and the tENO2 terminator.

6. The construction method of the modular orthogonal high-efficiency transcription factor DNA binding domain system according to claim 4, characterized in that, The sequence of the standard test vector constructed for E. coli is as shown in SEQ ID NO: 3, and the sequence of the standard test vector constructed for Saccharomyces cerevisiae is as shown in SEQ ID NO:

4.

7. The construction method of the modular orthogonal and highly efficient transcription factor DNA-binding domain system according to claim 1, wherein The reporter gene plasmid uses the yellow fluorescent protein YFP as the reporter gene in both the E. coli and Saccharomyces cerevisiae systems.

8. The method for constructing a modular orthogonal and highly efficient transcription factor DNA binding domain system according to claim 7, characterized in that, The strain is a Saccharomyces cerevisiae strain and an E. coli strain.

9. The construction method of the modular orthogonal and highly efficient transcription factor DNA binding domain system according to claim 1, wherein, The DNA binding domain and the operator sequence are obtained by mining the protein and operator sequence information in the database.

10. A modular orthogonal and efficient transcription factor DNA-binding domain system, characterized in that, Obtained by the construction method according to any one of claims 1-9.

Images