Method for constructing promoter library

Abstract

The invention belongs to the technical field of biotechnology, and specifically relates to a method for constructing a promoter library, which comprises: obtaining a genome sequence and a sequence annotation; setting promoter position information according to the sequence annotation; screening according to the promoter position information to obtain a promoter sequence to construct a promoter library; designing primers for one-step cloning connection according to the promoter sequence; obtaining promoter fragments according to the primers; constructing a recombinant vector according to the promoter fragments; transforming the recombinant vector and extracting a plasmid; and obtaining promoters of different strengths after the plasmid is transformed, thereby realizing promoter mining on a whole genome scale, and obtaining promoters of different strengths through virtual screening and experimental screening, and the construction method of the promoter library can also be applied to the mining and screening of natural promoters of other microorganisms.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and specifically relates to a method for constructing a promoter library. Background Art

[0002] Rhamnolipids (RL) is a kind of anionic biosurfactant of glycolipids, which has the functions of emulsification, solubilization, and reducing surface / interfacial tension. It is also low in toxicity and biodegradable. It can replace chemical surfactants to purify crude oil and achieve oil pollution remediation. It can also reduce the interfacial tension of oil reservoirs and improve oil recovery. In addition, RL can inhibit the growth of some bacteria and fungi, has good antibacterial activity, and has a good effect in promoting wound healing. RL has great application potential in the fields of oil extraction, medicine, food, daily chemicals and environmental protection due to its rich and excellent physical and chemical properties. In 2017, the report of Markets and Markets pointed out that the total market value of global biosurfactants has reached 4.2 billion US dollars and is expected to reach 5.52 billion US dollars in 2022. Therefore, the efficient and green synthesis of RL has important practical significance for promoting the rapid growth of the global economy.

[0003] Pseudomonas aeruginosa is currently the main bacterium producing rhamnolipids. This host contains a complete metabolic pathway for synthesizing rhamnolipids and is currently reported to have the highest rhamnolipid production. However, the expensive production and separation costs of rhamnolipids limit their industrial application. Studies have shown that when hydrophobic oils are used as carbon sources for fermentation, higher yields of rhamnolipids can be obtained, but their yield Y (P / S) (rhamnolipid concentration (g / L) / carbon source concentration (g / L)) is still at a low level (0.08-0.6). At the same time, due to the good emulsification properties of rhamnolipids, it is difficult to separate them from oils and fats, resulting in expensive separation costs. Therefore, it is crucial to increase the fermentation yield of rhamnolipids and reduce production and separation costs through reasonable metabolic engineering.

[0004] The in vivo synthesis mechanism of rhamnolipids is very complex. It is not only metabolically regulated by the three modules of sugar metabolism pathway, fatty acid synthesis pathway and rhamnosyltransferase cascade reaction, but also regulated by the group response mechanism. At present, most studies use a single gene knockout or gene overexpression method to increase RL production. In fact, the effect is minimal, because inappropriate gene knockout or overexpression will inhibit the metabolic balance of the bacteria themselves and thus affect RL production. Therefore, it is necessary to precisely fine-tune gene expression to study its impact on metabolic flow. As an important means to carry out metabolic engineering research, accurate "open-quantity" gene fine-tuning has a very broad application space and research value.

[0005] Promoter engineering is an important means to fine-tune metabolic flow control, including the mining of natural promoters, characterization of biological functional characteristics and artificial modification. It is an important basis for regulating gene expression and optimizing metabolite biosynthesis in metabolic engineering and synthetic biology research. Promoters are commonly used elements for regulating gene expression at the transcriptional level, including constitutive promoters, inducible promoters and specific promoters. Inducible promoters can regulate gene expression to a certain extent through induction time and inducer concentration, but this regulation can only control the start and stop of gene transcription. At the same time, the addition of inducers not only increases the cost of raw materials, but also increases the cost of metabolite separation and purification. Compared with inducible promoters, constitutive promoters do not require the addition of inducers, and their regulation is not affected by external conditions. The expression of the promoter gene is continuous, and the yield of metabolites between fermentation batches has good stability, which is more suitable for precise regulation of fermentation metabolic flow. However, there are very few promoter elements that can be used for Pseudomonas aeruginosa or other Pseudomonas, which makes it extremely difficult to use constitutive promoters to regulate the synthesis and metabolism of rhamnolipid in Pseudomonas aeruginosa. Therefore, it is crucial to develop a method for rapid screening and construction of promoter libraries.

[0006] Traditional promoter libraries are constructed mainly through error-prone PCR, saturation mutagenesis and promoter heterozygosity, using the strength of reporter genes to characterize the transcriptional strength of promoters. These strategies require a huge amount of screening to obtain mutant promoters with different regulatory strengths, which is not only time-consuming and labor-intensive, but also difficult to obtain ideal promoters. The development of bioinformatics and computer science has led to more and more attention and research on the rational design of promoter libraries.

[0007] Therefore, based on the above technical problems, it is necessary to design a new method for constructing a promoter library. Summary of the invention

[0008] The object of the present invention is to provide a method for constructing a promoter library.

[0009] In order to solve the above technical problems, the present invention provides a method for constructing a promoter library, comprising:

[0010] Obtain genome sequence and sequence annotation;

[0011] Set promoter position information based on sequence annotation;

[0012] Screening is performed according to the promoter position information to obtain promoter sequences to construct a promoter library;

[0013] Primers for one-step cloning ligation were designed based on the promoter sequence;

[0014] The promoter fragment is obtained according to the primers;

[0015] Constructing a recombinant vector based on the promoter fragment;

[0016] Transforming the recombinant vector and extracting the plasmid; and

[0017] After plasmid transformation, promoters with different strengths were obtained.

[0018] Further, the method for obtaining genome sequence and sequence annotation includes:

[0019] The genome sequence file and sequence annotation file of Pseudomonas aeruginosa PAO1.

[0020] Further, the method for setting promoter position information according to sequence annotation includes:

[0021] For the sense strand, the gene CDS start position −500 was used as the start position of the promoter;

[0022] For the antisense strand, the gene CDS start position +500 was used as the start position of the promoter.

[0023] Furthermore, the method of screening according to promoter position information to obtain promoter sequences to construct a promoter library includes:

[0024] Enter the promoter location information and promoter motif sequence:

[0025] TTGACANNNNNNNNNNNNNNNNNTATAAT;

[0026] The number of N is 15 to 18;

[0027] Set p value < 10 -6 , while ensuring sufficient screening volume, the promoter sequences corresponding to lower p values ​​were preferentially selected for the construction of promoter libraries.

[0028] Further, the method for obtaining a promoter fragment according to a primer comprises:

[0029] The promoter fragment was amplified by PCR using primers.

[0030] Further, the method for constructing a recombinant vector based on the promoter fragment comprises:

[0031] The promoter fragment was connected to the expression vector pUCP18E to construct a recombinant vector.

[0032] Further, the method for transforming the recombinant vector and extracting the plasmid comprises:

[0033] The recombinant vector was transformed into Escherichia coli and the plasmid was extracted.

[0034] Further, the method for obtaining promoters of different strengths after plasmid transformation includes:

[0035] The plasmids were sequentially transformed into Pseudomonas aeruginosa PAO1 competent cells, and promoters with different intensities were obtained by measuring the fluorescence intensity after fermentation culture.

[0036] Furthermore, the sequence of the primer is shown in any one of SEQ ID No. 11 to 30.

[0037] Furthermore, the promoter sequence is shown as SEQ ID No. 1-10.

[0038] The beneficial effects of the present invention are that the present invention obtains genome sequences and sequence annotations; sets promoter position information according to the sequence annotations; performs screening according to the promoter position information to obtain promoter sequences to construct promoter libraries; designs primers for one-step cloning connection according to the promoter sequences; obtains promoter fragments according to the primers; constructs recombinant vectors according to the promoter fragments; transforms the recombinant vectors and extracts plasmids; and obtains promoters of different strengths after the plasmids are transformed, thereby realizing promoter mining on a whole genome scale, and obtaining promoters of different strengths through virtual screening and experimental screening, and the construction method of the promoter library can also be applied to the mining and screening of natural promoters of other microorganisms; and constructs a promoter library based on bioinformatics and computer science methods, and adopts a method combining virtual screening with experimental screening, which can efficiently and quickly screen and mine promoters on the genome, meeting the needs of gene regulation research and engineering strain construction.

[0039] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or understood by practicing the present invention. The purpose and other advantages of the present invention are realized and obtained by the structures particularly pointed out in the description and the drawings.

[0040] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, preferred embodiments are given below and described in detail with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 is a flow chart of the method for constructing a promoter library of the present invention;

[0043] Figure 2 It is a schematic diagram of the PCR amplification result of the promoter gene of the present invention;

[0044] Figure 3 It is a schematic diagram of the colony PCR verification of the recombinant vector of the present invention;

[0045] Figure 4 It is a schematic diagram of measuring the fluorescence intensity of mCherry protein transcribed by different promoters of the present invention. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] like Figure 1 As shown, this embodiment provides a method for constructing a promoter library, including: obtaining a genome sequence and a sequence annotation; setting promoter position information according to the sequence annotation; screening according to the promoter position information to obtain a promoter sequence to construct a promoter library; designing primers for one-step cloning connection according to the promoter sequence; obtaining promoter fragments according to the primers; constructing a recombinant vector according to the promoter fragments; transforming the recombinant vector and extracting a plasmid; and obtaining promoters of different strengths after transforming the plasmid.

[0048] In this embodiment, the method for obtaining genome sequence and sequence annotation includes: a genome sequence file and a sequence annotation file of Pseudomonas aeruginosa PAO1.

[0049] In this embodiment, the method for setting promoter position information according to sequence annotation includes: for the positive strand, the gene CDS starting position -500 is used as the promoter starting position; for the antisense strand, the gene CDS starting position +500 is used as the promoter starting position.

[0050] In this embodiment, the method for screening according to promoter position information to obtain promoter sequences to construct a promoter library includes: inputting promoter position information, inputting promoter motif sequence: TTGACANNNNNNNNNNNNNNNNNNNNTATAAT; wherein the number of N is 15 to 18; setting p value <10 -6 , while ensuring sufficient screening volume, the promoter sequences corresponding to lower p values ​​were preferentially selected for the construction of promoter libraries.

[0051] In this embodiment, the method for obtaining a promoter fragment using primers includes: using primers to amplify the promoter fragment by PCR.

[0052] In this embodiment, the method for constructing a recombinant vector based on the promoter fragment includes: connecting the promoter fragment to the expression vector pUCP18E to construct a recombinant vector.

[0053] In this embodiment, the method for transforming the recombinant vector and extracting the plasmid includes: transforming the recombinant vector into Escherichia coli and extracting the plasmid.

[0054] In this embodiment, the method for obtaining promoters of different strengths after plasmid transformation includes: sequentially transforming plasmids into Pseudomonas aeruginosa PAO1 competent cells, and obtaining promoters of different strengths by measuring fluorescence intensity after fermentation culture.

[0055] In this embodiment, the sequence of the primer is shown in any one of SEQ ID No. 11-30.

[0056] In this example, the promoter sequences are shown in SEQ ID No. 1-10.

[0057] In this embodiment, the specific process can be: downloading the genome sequence file (.FASTA) and sequence annotation file (.GFF3) of Pseudomonas aeruginosa PAO1 from the NCBI database; using TB Tools to open the sequence file (.FASTA), manually inputting the position information of the promoter according to the sequence annotation file (.GFF3) to extract the promoter sequence (such as: for the positive chain, the gene CDS start position -500 is considered to be the start position of the promoter, and for the antisense chain, the gene CDS start position +500 is considered to be the start position of the promoter), and the software output file format is set to .FASTA format and named Extract.FASTA; using the motif prediction function of the FIMO module of the online software The MEME Suite, input the Extract.FASTA file, and input the promoter motif sequence According to the variability between the -35 region and the -10 region of the promoter, the number of N can be 15–18. In order to control the appropriate screening amount, the p value is set to <10 -6, under the premise of ensuring sufficient screening amount, the promoter sequence corresponding to the lower p value is preferentially selected for the construction of the promoter library, which is closer to the input promoter motif sequence and has higher conservation in the -35 region and the -10 region; according to the determined promoter sequence, the primers for one-step cloning connection are designed by software; the promoter fragment is amplified by PCR using the primers; the promoter fragment is connected to the expression vector pUCP18E to construct a recombinant vector; the recombinant vector is transformed into Escherichia coli and the plasmid is extracted; the plasmids are sequentially transformed into Pseudomonas aeruginosa PAO1 competent cells, and promoters of different intensities are obtained by measuring the fluorescence intensity after fermentation culture.

[0058] The present embodiment is further described below with reference to specific examples.

[0059] Extracting promoter sequences based on software

[0060] The chromosomal DNA sequence of Pseudomonas aeruginosa PAO1 was selected, and the genome sequence file (.FASTA) and sequence annotation file (.GFF3) of PAO1 were downloaded in turn. The sequence annotation file information can be viewed through Excel. Due to the huge amount of information of the entire genome sequence, only the promoter extraction method of the positive chain gene is used as an example to illustrate here. According to the CDS starting position of the annotated gene, the promoter region to be extracted is calculated and the position information of all promoter sequences is sorted out; open the TBtools software, click Fasta Tools in the "Sequence Toolkit" option, then select "FastaExtract", enter the above-sorted position information, set the file output name to Extract1.fasta, and click Start to generate the extraction file Extract1.fasta; use the motif prediction function of the FIMO module of the online software The MEME Suite (https: / / meme-suite.org / meme / index.html), the Extract1.fasta file in the above steps, enter the promoter motif sequence N is set to 18, and the p value is set to <10 in order to control the appropriate screening amount. -6 A total of 3000 gene promoter sequences were obtained through virtual screening. Due to the large number, we selected promoter sequences with a p value of 10 -7 The promoter sequences of 10 genes were named ParoE, PguaA, PppsA, PoprL, PamiE, PrpsJ, Prph, PbioB, PhupB, and PlexA, corresponding to sequences SEQID No.1 to 10, respectively. The -35 region and -10 region of their promoter sequences were closer to the input promoter motif sequence, and therefore they might be promoters with different promoter activities.

[0061] Acquisition of promoter fragment

[0062] 10 μL of Pseudomonas aeruginosa glycerol culture was inoculated into 30 mL of LB medium and cultured overnight at 37°C and 220 rpm. Then, 500 μL of the overnight culture was transferred to a 1.5 mL centrifuge tube and centrifuged at 12,000 rpm for 1 min to collect the bacteria. The Pseudomonas aeruginosa genomic DNA was extracted using a bacterial genome extraction kit.

[0063] The promoter fragment was amplified by PCR using the genome as a template. The reaction system was 50 μL. The 50 μL PCR system was: 1 μL genome, 2.5 μL upstream primer (10 μM), 2.5 μL downstream primer (10 μM), 0.5 μL 2× Phanta Master Mix high-fidelity DNA polymerase, and 32.5 μL ultrapure water. The primers were SEQ ID No. 11 to 30. The PCR program was: (1) 94°C, 10 min; (2) 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min; (3) 72°C, 10 min; (4) 16°C, +∞.

[0064] The results of promoter gene retrieval are as follows Figure 2 As shown (lanes 1-10 are: ParoE, PguaA, PppsA, PoprL, PamiE, PrpsJ, Prph, PbioB, PhupB, PlexA), all the above promoter gene fragments were obtained by amplification, and the fragment size was about 500bp. Afterwards, the DNA product gel recovery and purification kit of Shanghai Jierui Bioengineering Co., Ltd. was used to purify the promoter fragments obtained above, waiting for subsequent connection. The purification steps are detailed in its instructions.

[0065] Construction and verification of expression vector

[0066] In order to construct the recombinant plasmid, the plasmid pUCP18E-mCherry was double digested with Blp I and BamH I restriction endonucleases. The system was: 1 μg plasmid, 1 μL Blp I, 1 μL BamH I, 5 μL 10× buffer, ultrapure water to 50 μL, and digestion at 37°C for 30 min. After digestion, the vector fragment and the promoter fragment were connected using a cloning ligation kit from Nanjing Novozyme Biotechnology Co., Ltd. The connection system was: 60 ng of vector, a molar ratio of promoter fragment to vector fragment of 5-8:1, 2 μL 5×CE buffer, 1 μL ligase, and ultrapure water to 10 μL; the connection conditions were 37°C for 30 min. After that, the ligation product was transformed into E. coli BL21 (DE3) competent cells, cultured at 37°C for 1-1.5 h, and the transformation product was coated on an LB plate containing carbenicillin resistance and cultured overnight at 37°C.

[0067] The next day, a single colony was picked for colony PCR verification. The colony PCR system was as follows: pick one bacterium with a toothpick, 5 μL 2×Taq DNA polymerase, 0.5 μL M13F (10 μM), 0.5 μL M13R (10 μM), 0.5 μL dNTP (10 mM), and 4 μL ultrapure water. The sequence of M13F is: GTAAAACGACGGCCAGT; the sequence of M13R is: CAGGAAACAGCTATGAC. Colony PCR program: (1) 94°C, 10 min; (2) 30 cycles: 95°C, 30 s; 55°C, 30 s; 72°C, 1 min; (3) 72°C, 10 min; (4) 16°C, +∞. The colony PCR results are as follows: Figure 3 As shown (lanes 1-10 are: ParoE, PguaA, PppsA, PoprL, PamiE, PrpsJ, Prph, PbioB, PhupB, PlexA, respectively), since the pUCP18E-mCherry vector contains an mCherry band of about 713 bp, if the promoter is successfully connected to the vector, a fragment of about 1300 bp should be obtained. It can be seen from the figure that all promoters are successfully connected to the pUCP18E-mCherry plasmid.

[0068] For the positive transformants verified by PCR, single colonies were picked and inoculated into LB medium containing carbenicillin resistance, and cultured overnight at 37°C and 220rpm. The next day, an appropriate amount of bacterial solution was added with 30% glycerol and stored in a -80°C refrigerator. The remaining bacterial solution was used for plasmid extraction. For details on plasmid extraction, please refer to the instructions for the plasmid mini-extraction kit of Shanghai Jierui Bioengineering Co., Ltd. The plasmid was sent to Universal Biotechnology Co., Ltd. for sequencing verification.

[0069] Transformation of Pseudomonas aeruginosa PAO1 with expression vector

[0070] Take 3 μL of the verified recombinant vector and add it to the Pseudomonas aeruginosa PAO1 competent cells in turn, mix by pipetting, and incubate on ice for 30 minutes. Use the BIO-RAD Micro Pulser electroporator, select a 0.1 cm electric shock cup for transformation, the electric shock voltage is 1.6 kV, the time is 2.0 ms, and after the electric shock, place it on ice, quickly add 800 μL LB culture medium, pipette and resuspend, aspirate the bacterial solution and place it in a centrifuge tube, shake and culture in a 37 ° C shaker for 2 hours, then spread the inoculated solution on the LB plate containing carbenicillin resistance, take an appropriate amount of bacterial solution the next day, add 30% glycerol, and store it in a -80 ° C refrigerator.

[0071] Fluorescence intensity characterization of promoters

[0072] Take 5 μL of the recombinant Pseudomonas aeruginosa preserved in Example 4 and inoculate it into 30 mL LB medium. Culture it overnight at 37°C and 220 rpm. Pipette 1 mL of the bacterial solution into a 1.5 mL centrifuge tube and centrifuge it at 12000 rpm for 1 min. Remove the supernatant and resuspend the bacteria in each well with 200 μL of distilled water. After shaking and mixing, measure the OD on a Microplate reader. 600 The fluorescence intensity of the red fluorescent protein mCherry is 587nm, the maximum excitation wavelength is 610nm, and the data are collected to draw the fluorescence intensity diagram of mCherry transcribed by different promoters of Pseudomonas aeruginosa. The results are as follows Figure 4 As shown in the figure, it can be seen that constitutive promoters of Pseudomonas aeruginosa with different strengths were obtained through mining, and the strengths of these promoters are from high to low as PrpsJ>PoprL>PbioB>Prph>PlexA>PppsA>PamiE>PhupB>ParoE>PguaA, which proves the feasibility of the promoter library construction method.

[0073] In summary, the present invention obtains genome sequences and sequence annotations; sets promoter position information according to the sequence annotations; screens according to the promoter position information to obtain promoter sequences to construct promoter libraries; designs primers for one-step cloning connection according to the promoter sequences; obtains promoter fragments according to the primers; constructs recombinant vectors according to the promoter fragments; transforms the recombinant vectors and extracts plasmids; and obtains promoters of different strengths after the plasmids are transformed. The present invention realizes promoter mining on the whole genome scale by batch acquisition of natural promoters based on the Pseudomonas aeruginosa PAO1 genome sequence, and obtains promoters of different strengths through virtual screening and experimental screening. The construction method of the promoter library can also be applied to the mining and screening of natural promoters of other microorganisms. In addition, the promoter library is constructed based on the methods of bioinformatics and computer science, and the method combining virtual screening with experimental screening is adopted, so that the promoters on the genome can be efficiently and quickly screened and mined to meet the needs of gene regulation research and engineering strain construction.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. Inspired by the above ideal embodiment of the present invention, through the above description, relevant staff can make various changes and modifications without departing from the technical idea of ​​the present invention. The technical scope of this invention is not limited to the content in the specification, and its technical scope must be determined according to the scope of the claims.

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[0081] Sequence Listing <110> Changzhou Institute of Pharmaceutical Research Co., Ltd. <120> Method for constructing promoter library <130> 2021 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 500 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 1 acgtctaccc gcgctacaag gaatggtgcg accgctactt ccacctcaag caccgcaacg 60 agccgcgcgg catcggcggc ctgttcttcg acgacctcaa ccagtgggac ttcgacacct 120 gcttcgcctt catccgcgcc atcggcgatg cctatatcga cgcctacctg ccgatcgtcc 180 agcgccgcaa gcacacgccg ttcgatgaac ggcagcgaga gttccaggcc tatcgtcgcg 240 gtcgctatgt ggagttcaac ctggtcttcg atcgtggcac cctgttcggc ctgcagtccg 300 gcggccgtac cgaatcgatc ctgatgtcgt tgccgccgca ggtgcgctgg ggctatgact 360 ggaaacccga gccgggcagc gaggaagcgc gcctgaccga gtatttcctc gccgaccgcg 420 attggctcgc cggccagccc tgacccgccc gcaggggacg agtccgctcg tcccgtccca 480 ctccggcaaa ggaacgaccc 500 <210> 2 <211> 500 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 2 ggtcgccggc gcctactgtg tgatgatggg ctcgatgttc gccggtaccg aggaagcgcc 60 gggcgagatc gagctgttc agggccgttc ctacaagtc taccgcggca tgggttcgct 120 gggcgccatg tccggctccc agggctcgtc cgaccgctac ttccaggacg cctccgccgg 180 cgccgagaag ctggtgccgg aaggcatcga gggtcgcgtg ccgtacaagg gtgccctgtc 240 cgccatcgtc caccagctga tgggcggcct gcgcgccgcc atgggctaca ccggcagcgc 300 cgacatccag cagatgcgca cgcagccgca gttcgtccgg atcaccggcg cgggcatggc 360 cgagtcccat gtccacgacg tccagatcac caaggaagcc cccaactacc gggttggttg 420 aggctgattg aaatgagtgc tgcacggggc tgttcgattc agccccgtgt cgttttcgaa 480 ttccgtagcg agagatggcc 500 <210> 3 <211> 500 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 3 ccgaccgaat aggacgagtc ggccgaaagc tcctgttcca gaggcgaaag aaaggtggcg 60 aagatatcga tcatgaaacc gttggattgc gccaggaccg cccggatgtc gcggttcacg 120 atggtatcga agatgatcgg gcgggccccg tccgactcgg cagcattatt gatttgctgt 180 accatggcgc gcgctttttc ttcggtatcg atgtacggtc tcgtcagttt gacgaagctg 240 atgttctcga actgtgccag aaggctttgg cccagggttt cggcggtgat accggtgccg 300 tccgagatga agaatgcggt tcgtttcatt tgcaccgaaa gccttaaggt aggaacgatt 360 cttggctatg ataggccgcg cgttgtcaga ggcctttctg tcgcagcatt gtcacttatt 420 tctaagggcc aggccagaat ccagcggagc tgctcctggc cccgcttgag cttttccaat 480 tcaatagtgg agagatcacc 500 <210> 4 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 4 caacctggcg atcgataccg aacccttctg gggcaaggac ggctctaccc tgtacttcac 60 ctcggaccgt ggcggcaagc cgcagatcta caaaatgaac gtgaacagcg gtgccgtgga 120 ccgggtaacc ttcatcggca actacaacgc caacccgaaa ctctcggccg acgagaagac 180 cctggtaatg gtccaccgcc agcagggcta caccaacttc cagatcgccg cccaggacct 240 ccagcgcggc aatctgcggg tgctgtcgaa caccactctg gacgattcgc ccactgttgc 300 gcccaatggc accatgctaa tctacgccac ccgccagcag gaccggggcg tgctgatgct 360 cgtatccatc aacggacgcg tacggatacc tctccctacc gctcagggcg atgtgcgcga 420 gccttcctgg tccccttacc tgaactgacg gtcgccaacg gttacaaact tatacactgg 480 ggttcattag gagttacatg a 501 <210> 5 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 5 gccttgcaga cgctgtacgg cgggttgtcc ggtacgtccg ccggcgttgc cgaagtaggc 60 cgcgcaactg cgcttcgagg cgctgatgga gccgtcgttg cagaggaaga tatcgccgtc 120 gcaaccggcg ataccgcctt tctttcccga gcaggggcta ttggcggcca gcgccggaag 180 tgcgcagacc gacaggagga agagggcgat cgaggcgaag agtccgaatt tcattggcga 240 agcttccgtg cgaatgatgg ctgcatgcta tctcaggcgc gcgccgtgcg ctttcgcgag 300 cgcgccgatt acatggcctt gcccgagcct tgacagctcc ttccgacggg gcttataagt 360 ggcgccatca ggtcgtgcgc atcagcgtcg atgtcgcggg accgaaccta acgcatacgt 420 acagagcaaa tgggctctcc cggggttacc cgggagggcc ttttttcgtc ccgaaaaaat 480 aacaacaaga ggtgatatcc a 501 <210> 6 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 6 gcaccaagcg tgaagacgta gagcgtggcc aggtactggc caagccgggc accatcaagc 60 cgcacaccaa gttcgagtgc gaagtgtacg tgctgtccaa ggaagaaggt ggtcgtcaca 120 ccccgttctt caagggctac cgtccgcagt tctacttccg taccaccgat gtgaccggta 180 actgcgaact gccggaaggc gtagagatgg taatgccggg cgacaacatc aagatggttg 240 tcaccctgat cgctccgatc gccatggaag atggcctgcg cttcgcgatc cgcgaaggcg 300 gccgtaccgt tggcgccggc gtggttgcca agatcatcga gtaatcgctg atcctgccat 360 cagaaaaggg gccctccggg gccccttttt tcgttcgttg gttaaatatt gacaccctgt 420 gggggcatcc gtacaattgc gcctcctttt aacgggcgta gtccgtccgt tgggaatggt 480 aaattggagt ctgaggtcaa a 501 <210> 7 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 7 agcggatcga gcggcgccgg ctggcggatc agggcggcta cgccttcggc ggcggcgacc 60 agttggcggg cgcgctcctg gtcgacctgc aggctcttgc cggcggtttc ctcggcgaag 120 cgcagggtgc attccacctt gccacgcgac aggccctggc gcagcgcctc gcgcaccgcg 180 ccttcgaggt cgcggaaggc gtcgggcagg cgcaggtggg gctccaggta acggtggttg 240 accgagcgca gctcccagct cagggtgccg tgggtaccgg cctgctcgac gcgggcgaag 300 gcggtcatgc tgtgcaccat gggtctacct cgatagtcgt gtggggagtg cgttcggcgc 360 cggacgcgcg cggctgccgt gattcgagcc gggcattgta acccacgtcc ggcggggccc 420 gcacgccgac ggtggttgag agcccaggct ggcctctata atggcggcca attccgcttt 480 ctcagtacag gatatccacg a 501 <210> 8 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 8 acccgtatcg tgttttggaa agtggttggt cggaagggtc gaatatattt ttcgttatac 60 ttccgctgcg ctttgaatgg ctatatttag ggtggttggt tgcgttcatg gttcaagtct 120 ggataacttg ttttcgttgc gcaccgctag ggtggcaaga gattcaatgt ttctccatgt 180 tagaaacctg ccagggaata tataaataag atatttccta tttcttgtag gaaatatcct 240 aagcattact tgtagttttc cgaggtagtt gcgccgtttc ttctagggag ccgaatgcca 300 catagaggac gaggccgatt ggagcagtcg atgactactg gcttctacat gaatgatcaa 360 atattgatca gatgtagtcc tgtgtcctgg gttgggttga cagcgtcata cagccactta 420 tgatgccctt gagcctgacc cccggcgtgc cggggctccc cgacaaaagg cgccatacaa 480 gcgtcgaagg aatcttcccc a 501 <210> 9 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 9 gcatgtgcac ggcgctggtt tcggcgatca cgcagatccc ggtgcgcgcc gacgtggcaa 60 tgaccgggga gatcaccctg cgtgggcagg tgctggcgat cggcgggctg aaagaaaaac 120 ttttggcggc gcaccgcggc gggatcaaga cggtgatcat tcccgagga aatgttcgcg 180 acctgaagga aattccggac aatattaaga gtgatctggt tattaaccg gttaaatgga ttgacgaagt cctgcaaatt gcgctgcaat acgccccgga gcccttgccc gatgcggctc cggagatggt tgcaaaggat gaaaaacgcg agcctgattc caaggagcga attagcacgc 360 attagtcggt aggtattctt gacactgttt ttgggccctt gttataaagc ggcacttgct 420 gagtcagttg gcctaccagc gcagaatttc cttacaccta acacatagaa acatactcaa cagaaataag gggacttag g <210> 10 <211> 501 <212> DNA <213> Pseudomonas aeruginosa PAO1 <400> 10 aggaggtcct ccagggtggc gtgctgggcc tcggggcttgg cctggcgacg atccagctcc 60 ttttccaggc tggcgcagaa tggcccgagg aagcgcgaga acaccgcctg gatcagcgcc 120 ttcttcgaac cgaagtgata attcaccgcc gccaggttga ccccggcctt gctggtgatc 180 agcgcaacg aggtttcggc gaagcctttt tccgcgaaca gctgttccgc cgcatcgaga 240 atgcgttcga cggtttccga ctgggccatg gtttctccgc ctgacaaaca cttgtttgaa 300 acatacgttt cagccacatc ccttgtcaag cggcagatga cgcaccagtc aggaaaatta 360 cagagaagtc ccgaaaaacg gaagcaagac actggccaac ttcatcgtga cgcaggacga 420 aatgtcatgc ggaaaatcat tgccacgccc cgctcactgt atataatccc agtcactgga 480 taaaaacaca gagcgacgac a 501 <210> 11 <211> 42 <212> DNA <213> Artificial sequence <400> 11 gacggccagt gccaagctta gcacgtctac ccgcgctaca ag 42 <210> 12 <211> 39 <212> DNA <213> Artificial sequence <400> 12 acccttggag accatggatc cgggtcgttc ctttgccgg 39 <210> 13 <211> 40 <212> DNA <213> Artificial sequence <400> 13 gacggccagt gccaagctta gcggtcgccg gcgcctactg 40 <210> 14 <211> 40 <212> DNA <213> Artificial sequence <400> 14 acccttggag accatggatc cggccatctc tcgctacgga 40 <210> 15 <211> 43 <212> DNA <213> Artificial sequence <400> 15 gacggccagt gccaagctta gcccgaccga ataggacgag tcg 43 <210> 16 <211> 46 <212> DNA <213> Artificial sequence <400> 16 acccttggag accatggatc cggtgatctc tccactattg aattgg 46 <210> 17 <211> 42 <212> DNA <213> Artificial sequence <400> 17 gacggccagt gccaagctta gccaacctgg cgatcgatac cg 42 <210> 18 <211> 45 <212> DNA <213> Artificial sequence <400> 18 acccttggag accatggatc ctcatgtaac tcctaatgaa cccca 45 <210> 19 <211> 41 <212> DNA <213> Artificial sequence <400> 19 gacggccagt gccaagctta gcgccttgca gacgctgtac g 41 <210> 20 <211> 48 <212> DNA <213> Artificial sequence <400> 20 acccttggag accatggatc ctggatatca cctcttgttg ttattttt 48 <210> twenty one <211> 43 <212> DNA <213> Artificial sequence <400> twenty one gacggccagt gccaagctta gcgcaccaag cgtgaagacg tag 43 <210> twenty two <211> 46 <212> DNA <213> Artificial sequence <400> twenty two acccttggag accatggatc ctttgacctc agactccaat ttacca 46 <210> twenty three <211> 40 <212> DNA <213> Artificial sequence <400> twenty three gacggccagt gccaagctta gcagcggatc gagcggcgcc 40 <210> twenty four <211> 47 <212> DNA <213> Artificial sequence <400> twenty four acccttggag accatggatc ctcgtggata tcctgtactg agaaagc 47 <210> 25 <211> 44 <212> DNA <213> Artificial sequence <400> 25 gacggccagt gccaagctta gcacccgtat cgtgttttgg aaag 44 <210> 26 <211> 41 <212> DNA <213> Artificial sequence <400> 26 acccttggag accatggatc ctggggaaga ttccttcgac g 41 <210> 27 <211> 40 <212> DNA <213> Artificial sequence <400> 27 gacggccagt gccaagctta gcgcatgtgc acggcgctgg 40 <210> 28 <211> 48 <212> DNA <213> Artificial sequence <400> 28 acccttggag accatggatc cctctaagtc cccttatttc tgttgagt 48 <210> 29 <211> 41 <212> DNA <213> Artificial sequence <400> 29 gacggccagt gccaagctta gcaggaggtc ctccagggtg g 41 <210> 30 <211> 45 <212> DNA <213> Artificial sequence <400> 30 acccttggag accatggatc ctgtcgtcgc tctgtgtttt tatcc 45

Claims

1. A method for constructing a promoter library, characterized in that: include: Obtain genome sequence and sequence annotation; Set promoter position information based on sequence annotation; Screening is performed according to the promoter position information to obtain promoter sequences to construct a promoter library; Primers for one-step cloning ligation were designed based on the promoter sequence; The promoter fragment is obtained according to the primers; Constructing a recombinant vector based on the promoter fragment; Transform the recombinant vector and extract the plasmid; as well as After the plasmids were transformed, promoters of different strengths were obtained; The method for obtaining genome sequence and sequence annotation comprises: The genome sequence file and sequence annotation file of Pseudomonas aeruginosa PAO1; The method for setting promoter position information according to sequence annotation comprises: For the sense strand, the gene CDS start position −500 was used as the start position of the promoter; For the antisense strand, the gene CDS start position +500 was used as the promoter start position; The method of screening according to promoter position information to obtain promoter sequences to construct a promoter library comprises: Enter the promoter location information and promoter motif sequence: TTGACANNNNNNNNNNNNNNNNNTATAAT; The number of N is 15 to 18; Set p value < 10 -6 , while ensuring sufficient screening volume, the promoter sequences corresponding to lower p values ​​were preferentially selected for the construction of promoter libraries.

2. The method for constructing a promoter library according to claim 1, wherein The method for obtaining a promoter fragment according to a primer comprises: The promoter fragment was amplified by PCR using primers.

3. The method for constructing a promoter library according to claim 2, wherein: The method for constructing a recombinant vector based on a promoter fragment comprises: The promoter fragment was connected to the expression vector pUCP18E to construct a recombinant vector.

4. The method for constructing a promoter library according to claim 3, wherein: The method for transforming the recombinant vector and extracting the plasmid comprises: The recombinant vector was transformed into Escherichia coli and the plasmid was extracted.

5. The method for constructing a promoter library according to claim 4, characterized in that: The method for obtaining promoters of different strengths after plasmid transformation comprises: The plasmids were sequentially transformed into Pseudomonas aeruginosa PAO1 competent cells, and promoters with different intensities were obtained by measuring the fluorescence intensity after fermentation culture.

6. The method for constructing a promoter library according to claim 1, wherein: The sequence of the primer is shown in any one of SEQ ID No. 11-30.

7. The method for constructing a promoter library according to claim 1, wherein: The promoter sequences are shown in SEQ ID No. 1-10.

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