CN115678899A - A bidirectional promoter derived from Enterobacteriaceae and its application - Google Patents

Abstract

The invention relates to a bidirectional promoter derived from enterobacteria and its application, belonging to the field of genetic engineering. In order to find a bidirectional promoter that does not require the addition of exogenous inducers. The present invention provides a bidirectional promoter derived from Enterobacteriaceae, whose sequence is shown in SEQ ID NO.1 or SEQ ID NO.2, which can simultaneously promote the expression of genes from both positive and negative directions, and the positive and negative sides of the promoter The activation strength in the opposite direction is time-specific and is regulated by the growth period of the bacteria. The present invention is expected to provide a more favorable promoter element for the field of synthetic biology, and a promoter element for the field of fermentation without the need for inducer induction, and has broad application space and market prospects in the field of fermentation engineering.

Description

A bidirectional promoter derived from Enterobacteriaceae and its application

technical field

The invention belongs to the field of genetic engineering, and specifically relates to a bidirectional promoter derived from enterobacteriaceae and application thereof.

Background technique

Promoter is an important cis-element for gene expression regulation, which can effectively regulate the initiation, closure and abundance of downstream gene expression. Promoters are directional, and unidirectional promoters can specifically promote the expression of downstream genes. A bidirectional promoter refers to a DNA sequence located between two adjacent genes with opposite transcription directions, which can regulate gene transcription in both directions. Compared with traditional unidirectional promoters, bidirectional promoters can not only realize the expression of a single foreign gene, but also realize the simultaneous expression of two foreign genes in specific applications. Therefore, in the fields of synthetic biology, genetic engineering, etc. have wider application. The latest research shows that bidirectional promoters are widespread in eukaryotes and prokaryotes. However, only a few bidirectional promoters have been identified and used in transgenic plant and animal research. However, there are very few bidirectional promoters in bacterial systems of natural origin at present.

According to the temporal specificity, conditional specificity and spatial specificity of gene expression, promoters are further divided into constitutive promoters, tissue-specific promoters, and inducible promoters. Among them, the inducible promoter refers to that this type of promoter can greatly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals. The biggest advantage of inducible promoters is that they can quickly regulate gene expression or not, and are especially suitable for expressing toxic foreign proteins. Therefore, inducible promoters are widely used in the fields of transgenic animals and plants, genetic engineering strains, fermentation engineering, synthetic biology and medical research. For example, in the field of fermentation engineering, during the fermentation process, cells adapt to environmental changes and constantly adjust their own metabolic state, forming different stages such as lag phase, exponential growth phase, and product production phase. Flux distribution has different requirements. Therefore, it is widely used in fermentation culture to control the expression of the target gene at an appropriate time and obtain the target product. Currently, commonly used inducible promoters in bacteria include lac promoter (lactose promoter), trc and tac promoters (hybrid promoter of lactose and tryptophan), T7 phage promoter, L-arabinose-induced PBAD promoter, L-rhamnose-induced rha PBAD promoter, etc. However, this type of promoter generally requires expensive chemical reagents as an inducer, for example, an IPTG (Isopropylβ-D-Thiogalactoside) inducible promoter widely used in fermentation engineering, which is expensive The high IPTG has become an important factor in high cost in large-scale industrial fermentation. In addition to chemical reagent-induced promoters, there are also physical signal-induced promoters such as light-induced promoters and temperature-induced promoters. For large-scale fermentation systems, such promoters consume a lot of energy, and due to the long heating time, light is difficult to reach the fermentation system. Internally, the induced effect is not significant. Therefore, the development of self-inducible promoters that can induce the expression of target genes by relying on changes in the growth stage of the bacteria without the addition of exogenous inducers has great application prospects. However, no bidirectional sequential promoter derived from bacteria has been reported yet.

Contents of the invention

The purpose of the present invention is to search for a bidirectional promoter that does not require the addition of an exogenous inducer.

The present invention provides a bidirectional promoter derived from Enterobacteriaceae, the nucleotide sequence of the bidirectional promoter is shown in SEQ ID NO.1 or SEQ ID NO.2.

The invention provides a recombinant plasmid, the above-mentioned bidirectional promoter of the recombinant plasmid.

Further defined, the starting vector of the recombinant plasmid is pUC19, pACYCDuet-1, pETDuet1 or pTrcHis2b.

The present invention provides a recombinant bacterium, which contains the above-mentioned bidirectional promoter or the above-mentioned recombinant bacterium.

Further defined, the recombinant bacteria are Enterobacteriaceae or Escherichia coli as starting bacteria.

The invention provides a method for expressing genes without an inducer, using the above-mentioned recombinant bacteria to express genes.

It is further defined that the recombinant bacteria with an _OD600 of 4-6 are added to the reaction system in an amount of 1-5% by volume.

The present invention provides the application of the above-mentioned bidirectional promoter, the above-mentioned recombinant plasmid or the above-mentioned recombinant bacteria in strain fermentation.

Beneficial effects: the bidirectional promoter provided by the present invention can simultaneously activate the expression of genes from both positive and negative directions, and the activation strength of the positive and negative directions of the promoter has time specificity and is regulated by the growth period of the bacteria. The present invention is expected to provide a more favorable promoter element for the field of synthetic biology, and provide a promoter element for the field of fermentation that does not require inducer induction, and has broad application space and market prospects in the field of fermentation engineering.

Description of drawings

Figure 1 is a schematic diagram of plasmid pLUC.

Fig. 2 is a schematic diagram of plasmids pLUC-KAP1f and pLUC-KAP1r, wherein, A is a schematic diagram of pLUC-KAP1f, and B is a schematic diagram of pLUC-KAP1r.

Fig. 3 is a schematic diagram of plasmids pGFP-KAP1-LUC and pKan-GFP-KAP1-LUC, wherein, A is a schematic diagram of pGFP-KAP1-LUC, and B is a schematic diagram of pKan-GFP-KAP1-LUC.

Figure 4 is a diagram of the expression results of LUC initiated by the forward promoter KAP1f and reverse promoter KAP1r, wherein, A is the diagram of the expression results of LUC initiated by the reverse promoter KAP1r, and B is the diagram of the expression results of the forward promoter KAP1f; the abscissa is the group , and the ordinate is the LUC value.

Fig. 5 is the graph of the expression results of GFP and LUC initiated by the bidirectional promoter KAP1, wherein, A is the graph of the expression of LUC initiated by the bidirectional promoter KAP1, and B is the graph of the expression of GFP initiated by the bidirectional promoter KAP1 (RFU is the fluorescence intensity, which is proportional to the concentration of GFP Proportional, representing the concentration of GFP protein).

Fig. 6 is the time expression result of the genes on both sides of the bidirectional promoter KAP1, wherein A is the time expression result of the gene on the left side, and B is the time expression result of the gene on the right side.

Fig. 7 is the bacterial growth curve, wherein the abscissa is time, and the ordinate is OD ₆₀₀ .

specific embodiment

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the examples. The plasmid pLUC, competent cells, primers and reagents used in the examples can be purchased from commercial channels or obtained by conventional means well known to those skilled in the art.

Example 1. Discovery and functional verification of promoters

(1) Cloning of the promoter

Genome analysis of Enterobacter sp.CGMCC 5087 found that two genes were transcribed in the opposite direction, and the intergenic region was 185bp. Using the genome of Enterobacter sp.CGMCC 5087 (extracted using a microbial extraction kit) as a template, the 185bp DNA fragment was obtained by PCR with the primer pair KAP1-1/2, which was denoted as KAP1 (sequence shown in SEQ ID NO: 1). The 185bp DNA fragment, ie, the reverse promoter, was obtained by PCR with the primer pair KAP1-3/4, which was denoted as KAP1r (sequence shown in SEQ ID NO: 2).

(2) Construction of single reporter gene recombination vector

The construction of the pLUC vector: with the Pseudomonas syringae genome containing the luciferase gene as a template, the LUC (luxCDABE, sequence shown in SEQ ID NO:5) fragment is obtained by PCR with the primer pair LUC-1/2, and the fragment is Insert the BamHI and PstI sites of the pUC19 plasmid to obtain the LUC reporter vector pLUC. The map of the plasmid is shown in Figure 1.

Construction of pLUC-KAP1 recombinant vector: Insert the promoter fragments KAP1 and KAP1r obtained in step (1) into the BamHI site of pLUC plasmid respectively to obtain recombinant vectors pLUC-KAP1 and pLUC-KAP1r. The series of plasmid maps are shown in Figure 2 .

(3) Promoter activity verification

The recombinant vectors pLUC-KAP1 and pLUC-KAP1r were respectively transferred into the Escherichia coli DH5α strain, a single colony was picked and cultured overnight, and 150 μL of the bacterial solution was placed on a 96-well white plate, and placed in a microplate reader to detect the luminescence value. The test results are shown in Figure 4. The results proved that the KAP1 promoter has bidirectional promoter activity and can initiate the expression of genes on both sides

The OD ₆₀₀ of the bacterial liquid was detected by a spectrophotometer. The primer sequences are shown in Table 1.

Embodiment 2. A kind of construction method of the dual reporter gene recombination vector of verification promoter KAP1 bidirectional promoter activity, comprises the steps:

(1) Use Enterobacter sp.CGMCC 5087 genomic DNA as a template, and use KAP1-F and KAP1-R as primers to carry out PCR amplification to obtain the KAP1 promoter fragment; using the plasmid containing the GFP gene as a template, GFP-F, GFP- R is a primer for PCR amplification to obtain a GFP fragment (the gene sequence is shown in SEQ ID NO: 3); with GFP-F and KAP1-R as primers, GFP is fused with the fragment and the promoter fragment by fusion PCR to obtain GFP-KAP1 fusion fragment (sequence shown in SEQ ID NO: 4), GFP is located on the left side of the promoter.

(2) The GFP-KAP1 fragment was inserted into the EcoR I and BamH I sites of the pLUC plasmid to obtain a dual reporter gene recombinant vector pGFP-KAP1-LUC containing a bidirectional promoter KAP1. The plasmid map is shown in Figure 3A.

(3) With the primer pair Kan-1/2 and the plasmid pET28a as a template, the Kan gene fragment was obtained by PCR; the plasmid pGFP-KAP1-LUC was digested with BglI to obtain a linear fragment; the Kan fragment was connected to the linear plasmid to obtain Kan The resistant recombinant vector pKan-GFP-KAP1-LUC, the plasmid map is shown in Figure 3B.

The primer sequences are shown in Table 1.

Table 1 Primer sequence list

Functional verification of KAP1 as a bidirectional promoter:

(1) Transfer the recombinant vector pKan-GFP-KAP1-LUC into the Enterobacter sp.CGMCC5087 strain by electroporation (no need to verify KAP1r: both sides of the KAP1 promoter are connected to the reporter gene, so the forward and reverse directions can be directly detected promoter activity).

(2) Pick the strains stored at -80°C, streak on the LB plate for activation, and culture at 37°C for about 10-12 hours. At this time, a single colony grows, take out the plate, pick the bacteria with a toothpick or a white gun tip, and inoculate in LB Culture medium (with kanamycin added), cultivate overnight at 37°C, 180rpm.

(3) Take 150 μL of the bacterial solution and put it on a 96-well white plate, and put it in a microplate reader to detect the luminescence value, and the detection result is shown in Figure 5A. And detect OD ₆₀₀ with a spectrophotometer.

Result analysis: It shows that the promoter can start the expression of LUC reporter gene and has forward promoter activity.

(4) Take 1 mL of the bacterial liquid, collect the bacterial cells by centrifugation, wash once with ddH ₂ O, resuspend the bacterial cells with 1 mL of ddH ₂ O, take 150 μL of the bacterial liquid into a 96-well transparent plate (Costar 3635), and measure with a microplate reader GFP signal, the detection results are shown in Figure 5B. And detect OD ₆₀₀ with a spectrophotometer.

Result analysis: It shows that the promoter can initiate the expression of GFP reporter gene and has reverse promoter activity.

KAP1 promoter temporal function detection:

(1) (Wild-type Enterobacter sp.CGMCC 5087 strain, not transferred to any plasmid) strain activation: take the strains stored at -80°C, streak on the LB plate, cultivate overnight in a 37°C incubator, pick A single colony was inoculated in liquid LB medium, and the activated strain was cultured on a shaker at 37°C and 180 rpm.

(2) The cells were collected, washed twice with ddH ₂ O, resuspended with 1 mL of ddH ₂ O, transferred to 5 mL of M9 medium, adjusted to OD ₆₀₀ =0.1, and cultured on a shaker at 37° C. and 180 rpm.

(3) Samples were taken at 4h, 12h, 15h, 18h, 21h, and 24h, RNA was extracted, and gene expression on both sides of the KAP1 promoter was detected. The test results are shown in Figure 6. The OD ₆₀₀ was detected with a spectrophotometer to detect the growth of the bacteria, and the results are shown in FIG. 7 .

Analysis of the results: Under natural conditions, the KAP1 promoter can activate the expression of genes on both sides, and the expression of the gene on the left side increases with the growth of the bacteria, reaches the highest point when it reaches the stable phase, and then decreases; the gene on the right side is mainly in the logarithmic phase The expression decreased with the cell growth.

SEQUENCE LISTING

<110> Qingdao Institute of Bioenergy and Process Technology, Chinese Academy of Sciences

<120> A bidirectional promoter derived from Enterobacteriaceae and its application

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 185

<212>DNA

<213> Synthetic

<400> 1

gaacaggtat ccttctaatg ttgacctcac cttgagtatt aaataagcgt gatgactgtc 60

cagaattggc gataagaaga gcatggaaag aatgctttac aaaaaaggct gcgctatgct 120

gcttttttcc gcgttaatgt aaacgcatac actgatttttttcttcagcg ccagaggtca 180

tgaga 185

<210> 2

<211> 185

<212>DNA

<213> Synthetic

<400> 2

tctcatgacc tctggcgctg aagaaaaaaa tcagtgtatg cgtttacatt aacgcggaaa 60

aaagcagcat agcgcagcct tttttgtaaa gcattctttc catgctcttc ttatcgccaa 120

ttctggacag tcatcacgct tatttaatac tcaaggtgag gtcaacatta gaaggatacc 180

tgttc 185

<210> 3

<211> 717

<212>DNA

<213> Synthetic

<400> 3

ttatttgtag agctcatcca tgccatgtgt aatcccagca gcagttacaa actcaagaag 60

gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta 120

atggttgtct ggtgaaagga cagggccatc gccaattgga gtattttgtt gataatggtc 180

tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc 240

attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt 300

gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac 360

aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa 420

aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg 480

tttcatatga tctgggtatc ttgaaaagca ttgaacacca taagtcagag tagtgacaag 540

tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc 600

gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat taacgtcacc 660

atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcat 717

<210> 4

<211> 908

<212>DNA

<213> Synthetic

<400> 4

ttatttgtag agctcatcca tgccatgtgt aatcccagca gcagttacaa actcaagaag 60

gaccatgtgg tctctctttt cgttgggatc tttcgaaagg gcagattgtg tggacaggta 120

atggttgtct ggtgaaagga cagggccatc gccaattgga gtattttgtt gataatggtc 180

tgctagttga acgcttccat cttcaatgtt gtgtctaatt ttgaagttaa ctttgattcc 240

attcttttgt ttgtctgcca tgatgtatac attgtgtgag ttatagttgt attccaattt 300

gtgtccaaga atgtttccat cttctttaaa atcaatacct tttaactcga ttctattaac 360

aagggtatca ccttcaaact tgacttcagc acgtgtcttg tagttcccgt catctttgaa 420

aaatatagtt ctttcctgta cataaccttc gggcatggca ctcttgaaaa agtcatgctg 480

tttcatatga tctgggtatc ttgaaaagca ttgaacacca taagtcagag tagtgacaag 540

tgttggccat ggaacaggta gttttccagt agtgcaaata aatttaaggg taagttttcc 600

gtatgttgca tcaccttcac cctctccact gacagaaaat ttgtgcccat taacgtcacc 660

atctaattca acaagaattg ggacaactcc agtgaaaagt tcttctcctt tactcatctc 720

gaggaacagg tatccttcta atgttgacct caccttgagt attaaataag cgtgatgact 780

gtccagaatt ggcgataaga agagcatgga aagaatgctt tacaaaaaag gctgcgctat 840

gctgcttttt tccgcgttaa tgtaaacgca tacactgatt tttttcttca gcgccagagg 900

tcatgaga 908

<210> 5

<211> 5798

<212>DNA

<213> Synthetic

<400> 5

atgactaaaa aaatttcatt catttattaac ggccaggttg aaatctttcc cgaaagtgat 60

gatttagtgc aatccattaa ttttggtgat aatagtgttt acctgccaat attgaatgac 120

tctcatgtaa aaaacattat tgattgtaat ggaaataacg aattacggtt gcataacatt 180

gtcaattttc tctatacggt agggcaaaga tggaaaaatg aagaatactc aagacgcagg 240

acatacattc gtgacttaaa aaaatatatg ggatattcag aagaaatggc taagctagag 300

gccaattgga tatctatgat tttatgttct aaaggcggcc tttatgatgt tgtagaaaat 360

gaacttggtt ctcgccatat catggatgaa tggctacctc aggatgaaag ttatgttcgg 420

gcttttccga aaggtaaatc tgtacatctg ttggcaggta atgttccatt atctgggatc 480

atgtctatat tacgcgcaat tttaactaag aatcagtgta ttataaaaac atcgtcaacc 540

gatcctttta ccgctaatgc attagcgtta agttttattg atgtagaccc taatcatccg 600

ataacgcgct ctttatctgt tatatattgg ccccaccaag gtgatacatc actcgcaaaa 660

gaaattatgc gacatgcgga tgttattgtc gcttggggag ggccagatgc gattaattgg 720

gcggtagagc atgcgccatc ttatgctgat gtgattaaat ttggttctaa aaagagtctt 780

tgcattatcg ataatcctgt tgatttgacg tccgcagcga caggtgcggc tcatgatgtt 840

tgtttttacg atcagcgagc ttgtttttct gcccaaaaca tatattacat gggaaatcat 900

tatgaggaat ttaagttagc gttgatagaa aaacttaatc tatatgcgca tatattaccg 960

aatgccaaaa aagattttga tgaaaaggcg gcctattctt tagttcaaaa agaaagcttg 1020

tttgctggat taaaagtaga ggtggatatt catcaacgtt ggatgattat tgagtcaaat 1080

gcaggtgtgg aatttaatca accacttggc agatgtgtgt accttcatca cgtcgataat 1140

attgagcaaa tattgcctta tgttcaaaaa aataagacgc aaaccatatc tatttttcct 1200

tgggagtcat catttaaata tcgagatgcg ttagcattaa aaggtgcgga aaggattgta 1260

gaagcaggaa tgaataacat atttcgagtt ggtggatctc atgacggaat gagaccgttg 1320

caacgattag tgacatatat ttctcatgaa aggccatcta actatacggc taaggatgtt 1380

gcggttgaaa tagaacagac tcgattcctg gaagaagata agttccttgt atttgtccca 1440

taataggtaa aagtatggaa aatgaatcaa aatataaaac catcgaccac gttatttgtg 1500

ttgaaggaaa taaaaaaatt catgtttggg aaacgctgcc agaagaaaac agcccaaaga 1560

gaaagaatgc cattattatt gcgtctggtt ttgcccgcag gatggatcat tttgctggtc 1620

tggcggaata tttatcgcgg aatggatttc atgtgatccg ctatgattcg cttcaccacg 1680

ttggattgag ttcagggaca attgatgaat ttacaatgtc tataggaaag cagagcttgt 1740

tagcagtggt tgattggtta actacacgaa aaataaataa cttcggtatg ttggcttcaa 1800

gcttatctgc gcggatagct tatgcaagcc tatctgaaat caatgcttcg tttttaatca 1860

ccgcagtcgg tgttgttaac ttaagatatt ctcttgaaag agctttaggg tttgattatc 1920

tcagtctacc cattaatgaa ttgccggata atctagattt tgaaggccat aaattgggtg 1980

ctgaagtctt tgcgagagat tgtcttgatt ttggttggga agatttagct tctacaatta 2040

ataacatgat gtatcttgat ataccgttta ttgcttttac tgcaaataac gataattggg 2100

tcaagcaaga tgaagttatc aattgttat caaatattcg tagtaatcga tgcaagatat 2160

attctttgtt aggaagttcg catgacttga gtgaaaattt agtggtcctg cgcaattttt 2220

atcaatcggt tacgaaagcc gctatcgcga tggataatga tcatctggat attgatgttg 2280

atattactga accgtcattt gaacatttaa ctattgcgac agtcaatgaa cgccgaatga 2340

gaattgagat tgaaaatcaa gcaatttctc tgtcttaaaa tctattgaga tattctatca 2400

ctcaaatagc aatataagga ctctctatga aatttggaaa ctttttgctt acataccaac 2460

ctccccaatt ttctcaaaca gaggtaatga aacgtttggt taaattaggt cgcatctctg 2520

aggagtgtgg ttttgatacc gtatggttac tggagcatca tttcacggag tttggtttgc 2580

ttggtaaccc ttatgtcgct gctgcatatt tacttggcgc gactaaaaaa ttgaatgtag 2640

gaactgccgc tattgttctt cccacagccc atccagtacg ccaacttgaa gatgtgaatt 2700

tattggatca aatgtcaaaa ggacgatttc ggtttggtat ttgccgaggg ctttacaaca 2760

aggactttcg cgtattcggc acagatatga ataacagtcg cgccttagcg gaatgctggt 2820

acgggctgat aaagaatggc atgacagagg gatatatgga agctgataat gaacatatca 2880

agttccataa ggtaaaagta aaccccgcgg cgtatagcag aggtggcgca ccggtttatg 2940

tggtggctga atcagcttcg acgactgagt gggctgctca atttggccta ccgatgatat 3000

taagttggat tataaatact aacgaaaaga aagcacaact tgagctttat aatgaagtgg 3060

ctcaagaata tgggcacgat attcataata tcgaccattg cttatcatat ataacatctg 3120

tagatcatga ctcaattaaa gcgaaagaga tttgccggaa atttctgggg cattggtatg 3180

attcttatgt gaatgctacg actatttttg atgattcaga ccaaacaaga ggttatgatt 3240

tcaataaagg gcagtggcgt gactttgtat taaaaggaca taaagatact aatcgccgta 3300

ttgattacag ttacgaaatc aatcccgtgg gaacgccgca ggaatgtatt gacataattc 3360

aaaaagacat tgatgctaca ggaatatcaa atatttgttg tggatttgaa gctaatggaa 3420

cagtagacga aattattgct tccatgaagc tcttccagtc tgatgtcatg ccatttctta 3480

aagaaaaaca acgttcgcta ttatattagc taaggagaaa gaaatgaaat ttggattgtt 3540

cttccttaac ttcatcaatt caacaactgt tcaagaacaa agtatagttc gcatgcagga 3600

aataacggag tatgttgata agttgaattt tgaacagatt ttagtgtatg aaaatcattt 3660

ttcagataat ggtgttgtcg gcgctcctct gactgtttct ggttttctgc tcggtttaac 3720

agagaaaatt aaaattggtt cattaaatca catcattaca actcatcatc ctgtcgccat 3780

agcggaggaa gcttgcttat tggatcagtt aagtgaaggg agattattt tagggtttag 3840

tgattgcgaa aaaaaagatg aaatgcattt ttttaatcgc ccggttgaat atcaacagca 3900

actatttgaa gagtgttatg aaatcattaa cgatgcttta acaacaggct attgtaatcc 3960

agataacgat ttttatagct tccctaaaat atctgtaaat ccccatgctt atacgccagg 4020

cggacctcgg aaatatgtaa cagcaaccag tcatcatatt gttgagtggg cggccaaaaa 4080

aggtattcct ctcatcttta agtgggatga ttctaatgat gttagatatg aatatgctga 4140

aagatataaa gccgttgcgg ataaatatga cgttgaccta tcagagatag accatcagtt 4200

aatgatatta gttaactata acgaagatag taataaagct aaacaagaga cgcgtgcatt 4260

tattagtgat tatgttcttg aaatgcaccc taatgaaaat ttcgaaaata aacttgaaga 4320

aataattgca gaaaacgctg tcggaaatta tacggagtgt ataactgcgg ctaagttggc 4380

aattgaaaag tgtggtgcga aaagtgtatt gctgtccttt gaaccaatga atgatttgat 4440

gagccaaaaa aatgtaatca atattgttga tgataatatt aagaagtacc acatggaata 4500

tacctaatag atttcgagtt gcagcgaggc ggcaagtgaa cgaatcccca ggagcataga 4560

taactatgtg actggggtga gtgaaagcag ccaacaaagc agcagcttga aagatgaagg 4620

gtataaaaga gtatgacagc agtgctgcca tactttctaa tattatcttg aggagtaaaa 4680

caggtatgac ttcatatgtt gataaacaag aaattacagc aagctcagaa attgatgatt 4740

tgattttttc gagcgatcca ttagtgtggt cttacgacga gcaggaaaaa atcagaaaga 4800

aacttgtgct tgatgcattt cgtaatcatt ataaacattg tcgagaatat cgtcactact 4860

gtcaggcaca caaagtagat gacaatatta cggaaattga tgacatacct gtattcccaa 4920

catcggtttt taagtttact cgcttattaa cttctcagga aaacgagatt gaaagttggt 4980

ttaccagtag cggcacgaat ggtttaaaaa gtcaggtggc gcgtgacaga ttaagtattg 5040

agagactctt aggctctgtg agttatggca tgaaatatgt tggtagttgg tttgatcatc 5100

aaatagaatt agtcaatttg ggaccagata gatttaatgc tcataatatt tggtttaaat 5160

atgttatgag tttggtggaa ttgttatatc ctacgacatt taccgtaaca gaagaacgaa 5220

tagattttgt taaaacattg aatagtcttg aacgaataaa aaatcaaggg aaagatcttt 5280

gtcttattgg ttcgccatac tttatttatt tactctgcca ttatatgaaa gataaaaaaa 5340

tctcattttc tggagataaa agcctttata tcataaccgg aggcggctgg aaaagttacg 5400

aaaaagaatc tctgaaacgt gatgatttca atcatctttt atttgatact ttcaatctca 5460

gtgatattag tcagatccga gatatatta atcaagttga actcaacact tgtttctttg 5520

aggatgaaat gcagcgtaaa catgttccgc cgtgggtata tgcgcgagcg cttgatcctg 5580

aaacgttgaa acctgtacct gatggaacgc cggggttgat gagttatg gatgcgtcag 5640

caaccagtta tccagcattt attgttaccg atgatgtcgg gataattagc agagaatatg 5700

gtaagtatcc cggcgtgctc gttgaaattt tacgtcgcgt caatacgagg acgcagaaag 5760

ggtgtgcttt aagcttaacc gaagcgtttg atagttga 5798

<210> 6

<211> 40

<212>DNA

<213> Synthetic

<400> 6

cgtaatcatg gtcatctcga ggaacaggta tccttctaat 40

<210> 7

<211> 40

<212>DNA

<213> Synthetic

<400> 7

aattttttta gtcatggatc ctctcatgac ctctggcgct 40

<210> 8

<211> 40

<212>DNA

<213> Synthetic

<400> 8

cgtaatcatg gtcatctcga gtctcatgac ctctggcgct 40

<210> 9

<211> 40

<212>DNA

<213> Synthetic

<400> 9

aattttttta gtcatggatc cgaacaggta tccttctaat 40

<210> 10

<211> 79

<212>DNA

<213> Synthetic

<400> 10

gagctcggta cccggggatc catgactaaa aaaatttcac caagcttgca tgcctgcagt 60

caactatcaa acgcttcgg 79

<210> 11

<211> 38

<212>DNA

<213> Synthetic

<400> 11

gttcttctcc tttactcatg aacaggtatc cttctaat 38

<210> 12

<211> 28

<212>DNA

<213> Synthetic

<400> 12

aatggatcct ctcatgacct ctggcgct 28

<210> 13

<211> 30

<212>DNA

<213> Synthetic

<400> 13

aatgaattct tatttgtaga gctcatccat 30

<210> 14

<211> 38

<212>DNA

<213> Synthetic

<400> 14

attagaagga tacctgttca tgagtaaagg agaagaac 38

<210> 15

<211> 41

<212>DNA

<213> Synthetic

<400> 15

taaaccagcc agccggaagg gcgaagatcc tttgatcttt t 41

<210> 16

<211> 44

<212>DNA

<213> Synthetic

<400> 16

ccaacagttg cgcagcctga atggccaggt ggcacttttc gggg 44

Claims (8)

1. A bidirectional promoter derived from Enterobacteriaceae, characterized in that the nucleotide sequence of the bidirectional promoter is shown in SEQ ID NO.1 or SEQ ID NO.2. 2. A recombinant plasmid, characterized in that the recombinant plasmid contains the bidirectional promoter according to claim 1. 3. The recombinant plasmid according to claim 2, wherein the starting vector of the recombinant plasmid is pUC19, pACYCDuet-1, pETDuet1 or pTrcHis2b. 4. A recombinant bacterium, characterized in that the recombinant bacterium contains the bidirectional promoter according to claim 1 or the recombinant bacterium according to claim 2. 5. The recombinant bacterium according to claim 4, characterized in that the recombinant bacterium is based on Enterobacter or Escherichia coli. 6. A method for expressing genes without an inducer, characterized in that the recombinant bacteria according to claim 4 or claim 5 are used to express genes. 7. The method according to claim 6, characterized in that the recombinant bacteria with an _OD600 of 4-6 are added to the reaction system in an amount of 1-5% by volume. 8. The application of the bidirectional promoter according to claim 1, the recombinant plasmid according to claim 2 or 3, or the recombinant bacteria according to claim 4 or 5 in strain fermentation.

Images