CN108060174A - A kind of method for improving Escherichia coli Growth performance - Google Patents

A kind of method for improving Escherichia coli Growth performance Download PDF

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Publication number
CN108060174A
CN108060174A CN201810020498.6A CN201810020498A CN108060174A CN 108060174 A CN108060174 A CN 108060174A CN 201810020498 A CN201810020498 A CN 201810020498A CN 108060174 A CN108060174 A CN 108060174A
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escherichia coli
rpod
plasmid
growth performance
sequence
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王天文
靳会巧
刘紫薇
李萌
李一萌
罗欣琪
袁红雨
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Xinyang Normal University
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Xinyang Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

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  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention discloses a kind of method for improving Escherichia coli Growth performance, comprises the following steps：Using the genomic DNA of plant lactobacillus as template, with the coded sequence of PCR method amplification rpoD；Then, double digestion is carried out with Ncol and HindIII to PCR product and plasmid pET28a respectively, and cleaning treatment is carried out to the PCR product after double digestion, plasmid after double digestion is carried out to be tapped and recovered purifying, it is attached with T4 DNA ligases, recombinant plasmid is transformed into coli strain again, obtains the recombinant bacterial strain that can express RpoD transcription factors.The present invention promotes the growth performance of Escherichia coli, the coli strain is made to show advantage in growth, this is of great significance for the biotechnology research carried out based on this bacterial strain by introducing the controllable transcription factor gene that it is transcribed into Escherichia coli.

Description

A kind of method for improving Escherichia coli Growth performance
Technical field
The invention belongs to biological technical field, more particularly to a kind of method for improving Escherichia coli Growth performance.
Background technology
Escherichia coli have growth fast, and genetic manipulation is relatively easy, is sent out on a large scale using lower-cost matrix Ferment.Therefore, it is that the hosts of a variety of biological technology products is prepared in production, makes type strain in genetic engineering and at present The foreign protein prokaryotic expression system being most widely used.This system can produce a variety of biotechnology productions as " cell factory " Product.But the aspect that can improve there are many more it, such as its growth performance can further improve, to poor environment (such as generation Acid stress caused by thanking to the acetic acid of generation) resistance also have it is to be reinforced.Therefore, it is engineered and carry that effective gene is carried out to it The growth performance of high Escherichia coli, it has also become preferably utilize one of required solution during Escherichia coli progress biological product manufacture Major issue.
Transcription factor is a kind of functional protein molecule with special construction, energy controlling gene expression.It is controlled The intensity whether gene is transcribed and transcribes.Some transcription factors, by effectively identifying promoter, the expression of promotor gene, And this regulation and control are wide areas, and some transcription factors, then have very strong specific aim, and only the expression of portion gene is risen and is adjusted Control acts on.And the Function of cell as a whole, what to be intracellular all genes formed under transcription factor regulation and control answers The result of miscellaneous gene expression network interaction.If a transcription factor is wide area, then is regulated and controled by the transcription factor Number gene may be very big.Therefore, this transcription factor will also have an impact the multiple functions of cell.It is it means that logical Wide area transcription factor is overregulated, is expected to realize the improvement to cellular integrity energy.
The content of the invention
The shortcomings that it is an object of the invention to overcome the prior art and deficiency provide a kind of raising Escherichia coli Growth performance Method.
In order to achieve the above object, the present invention discloses following technical scheme：A kind of method for improving Escherichia coli Growth performance, It is characterised in that it includes following steps：
(1) using lactobacillus plantarum (Lactobacillus planttarum WSFC) genomic DNA as template, according to The sequence of transcription factor rpoD, the sequence of design primer rpoDf, rpoDr (add in NcoI restriction enzyme sites, in rpoDr in rpoDf Add in HindIII restriction enzyme sites), then the coded sequence with PCR method amplification rpoD；
(2) double digestion processing is carried out with NcoI and HindIII respectively to PCR product and plasmid pET28a, then it is distinguished It carries out being tapped and recovered purifying and PCR fragment cleaning treatment, be attached with T4-DNA ligases, complete recombinant plasmid PTE28a- The structure of rpoD；
(3) recombinant plasmid is imported into Escherichia coli.
As a preferred embodiment, recombinant plasmid is imported by e. coli bl21 using heat-shock transformed method in step (4) (DE3) culture experiment is carried out in and growth performance measures.
As a preferred embodiment, the primer for building pET28a-rpoD designs is as follows：
Sense primer rpoDf:CATGCCATGGAAATGGCAAAGGCAAAAGCAA
Anti-sense primer rpoDr:CCCAAGCTTTTATTCCAAGAAATCTTTTAACTGTTTACTACGT
As a preferred embodiment, 94 DEG C of -3min of cycling condition of rpoD genes are expanded in step (1), (94 DEG C of -20s, 60 DEG C of -15s, 72 DEG C of -1min10s) totally 30 cycle, 72 DEG C of -3min.
As a preferred embodiment, when carrying out double digestion in step (1) by double digestion system after mixing, place it in It in the PCR thin-wall tubes of 200 μ l, is suspended from beaker, it is made not contacted with the wall of beaker, be placed in that (frequency is in household microwave oven 2450MHz, power 800W) with " middle height " shelves heating 50s.
As a preferred embodiment, according to the sequence of transcription factor rpoD, design primer rpoDf and rpoDr in step (1) Sequence be respectively SEQ_ID No.2 and SEQ_ID No.3.
As a preferred embodiment, carrier used in step (1) is pET28a carriers.
As a preferred embodiment, double digestion selected in step (2) operation is quick enzymatic cleavage methods.
As a preferred embodiment, double digestion selected in step (2) operation is quick enzymatic cleavage methods, in domestic microwave Stove (frequency 2450MHz, power 800W) carries out in a manner that " middle height " shelves heat 50s.
As a preferred embodiment, recombinant plasmid is imported by bacillus coli DH 5 alpha using heat-shock transformed method in step (3) The middle screening and sequencing for carrying out correct recombinant plasmid.
As a preferred embodiment, recombinant plasmid is imported by e. coli bl21 using heat-shock transformed method in step (3) (DE3) culture experiment is carried out in and growth performance measures.
As a preferred embodiment, the culture of Escherichia coli carries out in culture medium LB.
As a preferred embodiment, it is by measuring OD in the incubation of Escherichia coli600To investigate Escherichia coli life Long performance.
The present invention effect be：By introducing the transcription factor gene from lactobacillus plantarum into Escherichia coli, realize To the integrally-regulated of its gene expression, and then the growth performance of Escherichia coli is improved, Escherichia coli are sent out in biological technical field Prior effect is waved, is of great significance.
Description of the drawings
Fig. 1：Recombinant plasmid pET28a-rpoD structure diagrams；
Fig. 2：Quick digestion operation chart；
Fig. 3：Recombinant bacterial strain E.coli containing recombinant plasmid pET28a-rpoD
The growth curve of BL21 (DE3)/pET28a-rpoD；
Fig. 4：The growth curve of control strain E.coli BL21 (DE3)/pET28a；
Specific embodiment
The sequence of transcription factor rpoD and recombinant plasmid pET28a-rpoD are respectively in SEQ_ID No.1 and SEQ_ID No.4 In provide, the protein sequence given expression to such as SEQ_ID No.5 are provided.A kind of side for improving Escherichia coli Growth performance of the present invention Method comprises the following steps：
1. using the genomic DNA of lactobacillus plantarum as template, according to the coded sequence of transcription factor rpoD, primer is designed RpoDf and rpoDr (add in Ncol restriction enzyme sites in sense primer rpoDf, HindIII digestions position are added in anti-sense primer rpoDr Point), with the coded sequence of PCR method amplification rpoD.The sequence of rpoD genes is as shown in SEQ_ID No.1；The sequence of rpoDf is such as Shown in SEQ_ID No.2；The sequence of rpoDr is as shown in SEQ_ID No.3.
2. carrying out double digestion with Ncol and HindIII to PCR product and plasmid vector pET28a respectively, tap rubber respectively Recovery purifying and PCR fragment cleaning treatment, are attached, construction recombination plasmid with T4DNA ligases.Recombinant plasmid pET28a- The sequence of rpoD is as shown in SEQ_IDNo.4, and the protein sequence given expression to is as shown in SEQ_ID No.5.
3. by the recombinant plasmid in step (2) through heat-shock transformed to escherichia coli DH5a competent cell.It is accredited as through PCR Positive transformant send Jin Sirui companies to carry out sequence verification.The correct recombinant plasmid of sequence, then it is transformed into e. coli bl21 (DE3) in bacterial strain, the recombinant bacterial strain of effable RpoD transcription factors is obtained.
With reference to case study on implementation, the present invention is further described, but should not be construed as limiting the invention；It is if not special It does not indicate, the conventional means that technological means used is well known to those skilled in the art in example.
A kind of embodiment 1, method for improving Escherichia coli Growth performance, comprises the following steps：1. with lactobacillus plantarum The genomic DNA of (Lactobacillus plantarum WSFC) is template, according to the coded sequence of transcription factor rpoD, if Primer rpoDf and rpoDr is counted (to add in Ncol restriction enzyme sites in sense primer rpoDf, HindIII is added in anti-sense primer rpoDr Restriction enzyme site) use PCR amplification rpoD coded sequence.The sequence of rpoD genes is as shown in SEQ_IDNo.1；The sequence of rpoDf is such as Shown in SEQ_ID No.2；The sequence of rpoDr is as shown in SEQ_IDNo.3.Shown in PCR system such as following table (1)；PCR cycle condition As shown in following table (2).
Table (1)：Expand the PCR system of rpoD genes
L.plantarum genomic DNAs 1μL
rpoDf(10uM) 2μL
rpoDr(10uM) 2μL
ExTaq(10U/μL) 1μL
10XExTaq buffer solutions 5μL
dNTP(10mM) 2μL
ddH2O 37μL
Table (2)：Expand the PCR cycle condition of rpoD genes
2. double digestion (digestion system such as following table is carried out with Ncol and HindIII to PCR product and plasmid pET28a respectively (3) shown in), it carries out being tapped and recovered purifying and PCR fragment cleaning treatment respectively, be attached with T4DNA ligases, structure restructuring Plasmid.The sequence of recombinant plasmid pET28a-rpoD is as shown in SEQ ID No.4.
Table (3)：The PCR product double digestion system of plasmid vector and rpoD
DNA (pET28a plasmids or PCR product) 1μg
NcoI(10U/μL) 1μL
HindIII(10U/μL) 1μL
10X buffer solutions H 5μL
ddH2O To 50 μ L
3. the operation of quick double digestion
By the component shown in above-mentioned table (3), mixed liquor is made, and is uniformly mixed.Place it in the PCR thin-wall tubes of 200 μ L In, it is suspended from so that it in beaker, to be made not contacted with the wall of beaker, beaker is placed in household microwave oven (frequency 2450MHz, work( Rate 800W), 50s is heated with " middle height " shelves, that is, completes double digestion.
4. by the recombinant plasmid in step (2), escherichia coli DH5a competent cell is transformed into through heat shock method, to through PCR Positive clone is accredited as, Jin Sirui companies is sent to carry out sequence verification.The correct recombinant plasmid reusable heat method of swashing is transformed into large intestine In bacillus BL21 (DE3) bacterial strain, the recombinant bacterial strain E.coli of effable RpoD transcription factors is obtained
BL21(DE3)/pET28a-rpoD。
5. with identical heat-shock transformed method, convert in plasmid pET28a to coli strain E.coliBL21 (DE3), shape Into E.coli BL21 (DE3)/pET28a (control strain).
A kind of embodiment 2, method for improving Escherichia coli Growth performance, comprises the following steps：
1. by control strain E.coli BL21 (DE3)/pET28a and the recombinant bacterial strain of expression transcription factor RpoD, difference In the flat lining outs of LB containing kanamycins (50mg/L), single bacterium colony is obtained；
2. inoculation single bacterium is dropped down onto in the test tube of the culture mediums of LB containing 10ml (kanamycins containing 50mg/L) respectively, in 37 DEG C of items Shaken cultivation stays overnight (200rpm) under part；
3. taking overnight culture 1ml, being inoculated into 50ml LB culture mediums respectively, (kanamycins containing 50mg/L and 0.01mM are different Propyl-β-D- thiogalactosides) in, 370C cultures；
4. being sampled every 30 minutes, its absorbance at 600nm is measured on spectrophotometer.It is bent to draw its growth Line, such as Fig. 3, shown in Fig. 4.
The above is only the preferred embodiments of the invention, it is noted that for those skilled in the art, On the premise of not departing from this clearly demarcated principle, it can also make and be suitably modified, these improvement also should be regarded as the protection model of the present invention It encloses.
Sequence table
<110>Xinyang Normal College
<120>A kind of method for improving Escherichia coli Growth performance
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1107
<212> DNA
<213> Lactobacillus plantarum WSFC
<400> 1
atggcaaagg caaaagcaac gacggaatac gacaaagccg ttaaagcatt aattaaggaa 60
tataagaaaa ctggtagcat tcaatatgat gaattatcag ataaattagc agcgccttac 120
aaactagatg ctagtggcat tgataaatta ttacaaaagg ttgaagatgc tgggattagt 180
gttgttgatg aaaaaggtga tcctgatgct cgagcggtca aaagtgtcaa aaaagtttct 240
aagaaggaac ttagtgacgc tggctcggct tctggaatta aaatcaatga tcccgtgcgg 300
atgtatttga aagaaattgg tcgtgtcgac ttattaacgg ctgacgaaga agtcgcttta 360
gcactccgaa tcgaacaagg tgatgaatct gccaagcaag aattagccga agccaactta 420
cggttggtcg tttcgattgc caaacgttac gtgggtcggg gaatgcaatt ccttgatttg 480
attcaagaag gtaacatggg gctaatgaag gccgttgaaa agtttgatta ccgtaaagga 540
ttcaagttct caacgtacgc gacttggtgg attcgccaag caattacgcg ggcaattgcc 600
gaccaagcgc ggaccattcg gattccagtg cacatggtcg aaactattaa caaattaatt 660
cgaattcaac ggcaactgtt acaagattta gggcgcgaac caactcctga agaaattggg 720
gctgaaatgg acatgccaac ggaaaaggtt cgtgaaatct tgaagatcgc acaagaaccc 780
gtttcattgg aaacgccaat tggtgaagag gacgattcac acttaggtga cttcatcgaa 840
gaccaagacg caacctcacc agcggatgcc gcggcttatg aattgttgaa agaacaactg 900
gaaggtgtcc ttgatacctt gacgaaccgg gaagaaaacg tcttacgctt acggttcgga 960
cttgatgatg gtcggacccg gacactggaa gaagttggaa aagtcttcgg tgtgacccgt 1020
gaacggatcc gccaaatcga agccaaggcg ctacggaagt tacgccaccc atcacgtagt 1080
aaacagttaa aagatttctt ggaataa 1107
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<213> Lactobacillus plantarum WSFC
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catgccatgg aaatggcaaa ggcaaaagca a 31
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<213> Lactobacillus plantarum WSFC
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cccaagcttt tattccaaga aatcttttaa ctgtttacta cgt 43
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<213> Lactobacillus plantarum WSFC
<400> 4
atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa 60
ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt 120
tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagctttt 180
attccaagaa atcttttaac tgtttactac gtgatgggtg gcgtaacttc cgtagcgcct 240
tggcttcgat ttggcggatc cgttcacggg tcacaccgaa gacttttcca acttcttcca 300
gtgtccgggt ccgaccatca tcaagtccga accgtaagcg taagacgttt tcttcccggt 360
tcgtcaaggt atcaaggaca ccttccagtt gttctttcaa caattcataa gccgcggcat 420
ccgctggtga ggttgcgtct tggtcttcga tgaagtcacc taagtgtgaa tcgtcctctt 480
caccaattgg cgtttccaat gaaacgggtt cttgtgcgat cttcaagatt tcacgaacct 540
tttccgttgg catgtccatt tcagccccaa tttcttcagg agttggttcg cgccctaaat 600
cttgtaacag ttgccgttga attcgaatta atttgttaat agtttcgacc atgtgcactg 660
gaatccgaat ggtccgcgct tggtcggcaa ttgcccgcgt aattgcttgg cgaatccacc 720
aagtcgcgta cgttgagaac ttgaatcctt tacggtaatc aaacttttca acggccttca 780
ttagccccat gttaccttct tgaatcaaat caaggaattg cattccccga cccacgtaac 840
gtttggcaat cgaaacgacc aaccgtaagt tggcttcggc taattcttgc ttggcagatt 900
catcaccttg ttcgattcgg agtgctaaag cgacttcttc gtcagccgtt aataagtcga 960
cacgaccaat ttctttcaaa tacatccgca cgggatcatt gattttaatt ccagaagccg 1020
agccagcgtc actaagttcc ttcttagaaa cttttttgac acttttgacc gctcgagcat 1080
caggatcacc tttttcatca acaacactaa tcccagcatc ttcaaccttt tgtaataatt 1140
tatcaatgcc actagcatct agtttgtaag gcgctgctaa tttatctgat aattcatcat 1200
attgaatgct accagttttc ttatattcct taattaatgc tttaacggct ttgtcgtatt 1260
ccgtcgttgc ttttgccttt gccatttcca tggtatatct ccttcttaaa gttaaacaaa 1320
attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag tcgtattaat 1380
ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc atcaccggcg 1440
ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa gatcgggctc 1500
gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg 1560
ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg gtgctcaacg 1620
gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga gagcgtcgag 1680
atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata gcgcccggaa 1740
gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat 1800
gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg 1860
aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg 1920
gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc 1980
ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc 2040
agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac 2100
aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat 2160
gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac 2220
cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag 2280
catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc 2340
tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg 2400
atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg 2460
ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc 2520
gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga 2580
tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat 2640
tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg 2700
gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc 2760
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 2820
gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt agctcactca 2880
ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca gctccttccg 2940
gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta tcatgcaact 3000
cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct ttcgctggag 3060
cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc 3120
cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta tcgccggcat 3180
ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc taggctggcg 3240
gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt gaagcgactg 3300
ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt tccgtgtttc 3360
gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga tctgcatcgc 3420
aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc gctggcattg 3480
accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt taccctcaca 3540
acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca tcctctctcg 3600
tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg catcagtgac 3660
caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga cattaacgct 3720
tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg aatcgcttca 3780
cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg acggtgaaaa 3840
cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 3900
cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac 3960
ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt 4020
gtactgagag tgcaccatat atgcggtgtg aaataccgca cagatgcgta aggagaaaat 4080
accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc 4140
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg 4200
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg 4260
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac 4320
gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 4380
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 4440
ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg 4500
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct 4560
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac 4620
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt 4680
tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 4740
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 4800
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat 4860
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac 4920
gttaagggat tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg 4980
ggtgttatga gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac 5040
atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 5100
acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 5160
ggtagcgttg ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt 5220
atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 5280
actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 5340
aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 5400
tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 5460
ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 5520
tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat 5580
ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 5640
cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 5700
ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 5760
aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg 5820
atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg 5880
aaaagtgcca cctgaaattg taaacgttaa tattttgtta aaattcgcgt taaatttttg 5940
ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa 6000
agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa 6060
gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg 6120
tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa 6180
ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa 6240
ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct 6300
gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc 6360
a 6361

Claims (6)

  1. A kind of 1. method for improving Escherichia coli Growth performance, which is characterized in that comprise the following steps：
    (1) growth performance of Escherichia coli is improved by introducing transcription factor, selected transcription factor is from plant breast bar The transcription factor rpoD of bacterium (Lactobacillus plantarum WSFC), sequence is as shown in SEQ_ID NO.1；Use PCR Method expands the coded sequence of rpoD；
    (2) construction recombination plasmid：Respectively to PCR product and plasmid with carrying out double digestion, and the PCR product after double digestion is carried out Cleaning treatment carries out the plasmid after double digestion to be tapped and recovered purifying, is attached with T4DNA ligases；
    (3) recombinant plasmid is transformed into coli strain again, obtains the recombinant bacterial strain that can express RpoD transcription factors.
  2. 2. a kind of method for improving Escherichia coli Growth performance according to claim 1, which is characterized in that in step (1) Include procedure below with the coded sequence of PCR method amplification rpoD:
    (1) using lactobacillus plantarum (Lactobacillus planttarum WSFC), genomic DNA is as template, according to transcription The sequence of factor rpoD, the sequence of design primer rpoDf, rpoDr, the sequence of primer rpoDf, rpoDr are respectively such as SEQ_ID Shown in No.2 and SEQ_ID No.3.
  3. A kind of 3. method for improving Escherichia coli Growth performance according to claim 1 or 2, which is characterized in that step (2) Middle construction recombination plasmid includes procedure below:
    Double digestion processing is carried out with NcoI and HindIII respectively to PCR product and plasmid pET28a, then is tapped rubber respectively to it Recovery purifying and PCR fragment cleaning treatment, are attached with T4-DNA ligases, complete the structure of recombinant plasmid pET28a-rpoD It builds.
  4. A kind of 4. method for improving Escherichia coli Growth performance according to claim 1, which is characterized in that the weight after connection Group plasmid is transferred to bacillus coli DH 5 alpha and carries out the measure of sequence.
  5. A kind of 5. method for improving Escherichia coli Growth performance according to claim 1, which is characterized in that correctly restructuring Plasmid enters Escherichia coli by conversion method, and is cultivated in LB culture mediums.
  6. 6. a kind of method for improving Escherichia coli Growth performance according to claim 1, which is characterized in that measure bacterium solution and exist OD600The absorbance at place investigates facilitation of the transcription factor to growth.
CN201810020498.6A 2018-01-10 2018-01-10 A kind of method for improving Escherichia coli Growth performance Pending CN108060174A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116694657A (en) * 2023-06-06 2023-09-05 江南大学 Method for changing cell growth and metabolic synthesis of escherichia coli based on sigma factor regulation

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN105238727A (en) * 2015-11-13 2016-01-13 江南大学 Lactobacillus plantarum capable of antagonizing campylobacter jejuni and inhibiting expression of flaA gene of campylobacter jejuni

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CN105238727A (en) * 2015-11-13 2016-01-13 江南大学 Lactobacillus plantarum capable of antagonizing campylobacter jejuni and inhibiting expression of flaA gene of campylobacter jejuni

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STEFAN M. GAIDA等: "Expression of heterologous sigma factors enables functional screening of metagenomic and heterologous genomic libraries", 《NATURE COMMUNICATIONS》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116694657A (en) * 2023-06-06 2023-09-05 江南大学 Method for changing cell growth and metabolic synthesis of escherichia coli based on sigma factor regulation

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