CN106480082A - A kind of gene knockout method for pseudomonas putida NBRC 14164 - Google Patents
A kind of gene knockout method for pseudomonas putida NBRC 14164 Download PDFInfo
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- CN106480082A CN106480082A CN201610983580.XA CN201610983580A CN106480082A CN 106480082 A CN106480082 A CN 106480082A CN 201610983580 A CN201610983580 A CN 201610983580A CN 106480082 A CN106480082 A CN 106480082A
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Abstract
The present invention is a kind of gene knockout method for pseudomonas putida NBRC 14164, build gene knockout plasmid first, search and intend knocking out gene order, after designing gene knockout primer and knocking out, verify primer, amplified production successfully constructs through enzyme action, connection, plasmid；Then according to the preparation of heat-shock transformed method engages the supplementary strain competent escherichia coli cell of conversion, whole strict aseptic technique, then by heat-shock transformed for gene knockout plasmid entrance competent escherichia coli cell；Finally import plasmid to P.putida using joint conversion method, engage trans-utilization twice homologous recombination by gene knockout, first time homologous recombination plasmid enters P.putida from E.coli and is incorporated on chromosome, second homologous recombination plasmid is departed from from chromosome with the gene knocking out primer amplification, i.e. gene knockout success.
Description
Technical field
The present invention relates to a kind of gene knockout method for pseudomonas putida NBRC 14164, it belongs to molecular biology
Research field.
Background technology
Pseudomonas putida (Pseudomonas putida) is a class gram negative bacteria, belongs to γ-mycetozoan, extensively divides
It is distributed in the Ecological niche of nature, due to its powerful metabolic capacity, environment is had compared with strong adaptability, such as can survive in high concentration
In the environment such as heavy metal pollution or organic pollution [1,2,3].With the development of sequencing technologies, many typical case's P.putida bacterial strains
Genome sequence inquired about, be that the extensive application of bacterial strain, genetic background parsing, molecular modification etc. provide strong
Support.As great representational P.putida KT2440 has good behaviour in terms of PHA produces [4], and the generation of its genome dimension
The structure thanking to network model more allows and it greatly improves PHA yield [5].
At present, gene knockout method has a lot, is wherein had widely using the technology that homologous recombination principle developed
Application.Homologous recombination refers to the property utilizing living cells DNA that restructuring can occur in genetic engineering with exogenous DNA array, to carry out
The technology [6] of a certain genes of interest in pointed decoration modifying gene group, can be divided into induced gene to knock out method, gene insertion mutation
Technology, suicide vector constructing technology etc..Wherein, the principle of suicide vector is：The genes of interest that need are lacked is cloned into suicide and carries
On body, make it into host by engaging etc., due to there is not replication protein needed for replicator initial in Host Strains
Cannot replicate, in the presence of extraneous selection pressure, mutant gene entrained by suicide vector just with host chromosome on
There is secondary homologous recombination in wild type gene, create the mutant with mutational site, and carrier itself is due to Suicide spy
Property pass in thalline disappearance [7] together with wild type gene original on chromosome with antibacterial.
The whole genome sequence of P.putida NBRC 14164 measured [3] in 2014, in DDBJ/EMBL/GenBank
It is AP013070 etc. the numbering in multiple data bases.Before this, the relative analyses during this bacterial strain is only used as studying, are such as used for dividing
Class technique study [8], toxic pollutant degraded [9] etc.；Even to this day, still rarely has the report specialized in for this bacterial strain.
Explore the operability of P.putida NBRC 14164 from molecular level, a kind of operational approach that can imitate of row is provided, will be it
New passage is opened in further investigation, opens up wide space.
[1]Wu X,Monchy S,Taghavi S,et al.,Comparative genomics and functional
analysis of niche-specific adaptation in Pseudomonas putida.FEMS Microbiol.,
35:299-323(2011).
[2]Poblete-Castro I,Becker J,Dohnt K,et al.,Industrial biotechnology
of Pseudomonas putida and related species.Appl.Microbiol.Biotechnol.,93:2279-
2290(2012).
[3]Shoko O,Atsushi Y,Akira H,et al.,The complete genome sequence of
Pseudomonas putida NBRC 14164Tconfirms high intraspecies variation.Genome
Announcements,2(1):e00029-14(2014).
[4]Wang Q,Nomura CT,Monitoring differences in gene expression levels
and polyhydroxyalkanoate(PHA)production in Pseudomonas putida KT2440grown on
different carbon sources.Journal of Bioscience&Bioengineering,110(6):653-659
(2010).
[5]Nogales J,PalssonThiele I,A genome-scale metabolic
reconstruction of Pseudomonas putida KT2440:iJN746as a cell factory.BMC
Systems Biology,2(37):1-20(2008).
[6] congratulating floods, simple and clear Principles of Gene Engineering [M]. Bei Ai Science Press, 2005:389.
[7] Lu Fuzhi, Sun Jing, Huang Jinghua, etc., the structure of plasmid pUC19-CM-D and application. Agriculture of Anhui science, 38
(19):9953-9954(2010).
[8]Tamura H,Hotta Y,Sato H,Novel accurate bacterial discrimination by
MALDI-time-of-flight MS based on ribosomal proteins coding in S10-spc-alpha
operon at strain level S10-GERMS.Journal of the American Society for Mass
Spectrometry,24(8):1185-93(2013).
[9]Nonaka K,Ohta H,Sato Y,et al.,Utilization of phenylpropanoids by
Pseudomonas putida soil isolates and its probable taxonomic
significance.Microbes&Environments,23(4):360-364(2008).
Content of the invention
In view of above-mentioned, this proposes a kind of gene knockout method for pseudomonas putida NBRC 14164, and verifies
Its feasibility, particular technique is as follows：
Build gene knockout plasmid first, the supplementary strain E.coli S17-1 competence then preparing joint conversion is thin
Born of the same parents, then by gene knockout plasmid Transformed E .coli S17-1 competent cell, finally import plasmid extremely using joint conversion method
In P.putida NBRC 14164, electrophoresis is verified.
It is described as follows：
Pseudomonas putida Pseudomonas putida NBRC 14164, buys and protects from Chinese industrial microorganism fungus kind
Hide administrative center.Angstrom Xi Shi escherichia coli Escherichia coli S17-1 helps out in engaging conversion, with carrier
Plasmid pK18mobSacB is Laboratories Accession (original strain is purchased from Wuhan Miao Ling bio tech ltd).
A kind of gene knockout method for pseudomonas putida NBRC 14164, its step is as follows：
1) gene knockout plasmid construction；
2) preparation of E.coli S17-1 competent cell；
3) gene knockout plasmid Transformed E .coli S17-1 competent cell；
4) engage conversion method and import plasmid to P.putida NBRC 14164.
Described step 1) be：Search and intend knocking out gene order, design clpp gene using software Primer Premier 5
Verify primer except primer and after knocking out, two pairs of primer extension products differ 300-400bp, knock out primer extension product and connect through enzyme action
Connect plasmid to successfully construct.
Described step 2) be：Supplementary strain E.coli for engaging conversion is thin according to heat-shock transformed method preparation competence
Born of the same parents, use Mg2+Solution, Ca2+The washing of solution, whole sterile working.
Described step 3) be：With heat-shock transformed method, gene knockout plasmid is converted and enter E.coli competent cell.
Described step 4) be：Engage trans-utilization twice homologous recombination by gene knockout, first time homologous recombination plasmid
Enter P.putida from E.coli and be incorporated on chromosome, the gene of second homologous recombination plasmid and knockout primer amplification
Depart from from chromosome, i.e. gene knockout success.
First time homologous recombination condition be pseudomonas putida with the escherichia coli containing restructuring suicide vector plasmid in 30
DEG C, 200rpm cultivates 12h, then stands 12h.
Second homologous recombination condition is that the successful single bacterium colony of first time homologous recombination after screening is inoculated into non-resistant LB
In fluid medium, 30 DEG C, 200rpm cultivates 24h.
It is an advantage of the invention that：Using engaging method for transformation, using homologous recombination principle, by supplementary strain E.coli
S17-1, suicide vector plasmid pK18mobSacB complete gene knockout jointly.Explore P.putida NBRC from molecular level first
14164 operability, provides a kind of operational approach that can imitate of row；The method is applicable not only to gene knockout, also can outward
Source gene integration, to P.putida chromosome, imports compared to exogenous plasmid and has more preferable stability.The method will be
New passage is opened in the further investigation of P.putida NBRC 14164, opens up wide space.
Brief description
Fig. 1 enzyme action connection diagram
Gene to be knocked out first expands from P.putida genome, and the upstream and downstream primer being wherein used for amplification contains enzyme
Enzyme site.By the gene after amplification with suicide vector plasmid with, after identical restriction enzyme cleavage, getting final product success through connecting
Build gene knockout plasmid.
Fig. 2 competent cell prepares schematic diagram
For engaging the supplementary strain E.coli of conversion through Mg2+Solution, Ca2+The washing of solution, finally uses glycerite weight
Outstanding can becoming is applied to heat-shock transformed competent cell.
The heat-shock transformed schematic diagram of Fig. 3
Soft dot represents suicide vector plasmid, and through heat shock and ice bath, suicide vector plasmid passes through the cell wall of E.coli
Enter in cell with cell membrane.
Fig. 4 gene knockout schematic diagram
On carrier, gray fragment is to intend knocking out genetic fragment, and on genome, greyish black other slice is complete genome；For the first time
After homologous recombination, suicide vector plasmid integration is to genome；After second homologous recombination, suicide vector plasmid departs from genome,
Remaining black fragment is genetic fragment after knockout.
Electrophoretogram before and after Fig. 5 prs and rpiA gene knockout
Swimming lane 3 is DNA molecular amount standard, and swimming lane 1 and 4 is respectively prs the and rpiA gene before knocking out, and swimming lane 2 and 5 is respectively
For prs the and rpiA gene after knocking out.
Specific embodiment
Below in conjunction with the accompanying drawings and specific embodiment, the present invention is described in further detail：
1. culture medium prescription and solution formula
SOB culture medium：Tryptone 20g/L, yeast powder 5g/L, sodium chloride 0.5g/L, 250mM potassium chloride 10mL/L, 2M
Magnesium chloride 5mL/L；
LB culture medium：Peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L；
LBS culture medium：Peptone 10g/L, yeast powder 5g/L, sodium chloride 10g/L, sucrose 60g/L；
Prepare and during solid medium, add agar powder 15g/L；
Ampicillin (Amp) working concentration：100μg/mL；
Kanamycin (Kan) working concentration：50μg/mL；
Mg2+Solution：100mM magnesium chloride, 10mM TriS, pH7.5；
Ca2+Solution：100mM calcium chloride, 10mM TriS, pH7.5；
Glycerite：8.5mL Ca2+Solution, 1.5mL glycerol.
2. gene knockout plasmid construction
(1) search the complete sequence of P.putida gene in ncbi database, taking prs and rpiA as a example design gene knockout
Verify that primer is as shown in table 1 after primer and knockout.Wherein checking primer extension product is about full length gene, knocks out primer amplification and produces
Thing is about full length gene and removes head and the tail 100-200bp, and that is, two pairs of primer extension products differ 200-400bp.
Primer is verified after table 1prs and rpiA gene knockout primer and knockout
(2) primer amplification gene is knocked out for template with P.putida glycerol stock, electrophoresis is verified correct amplified production
With sequence two ends identical restriction enzyme cleavage 1h of pK18mobSacB, be connected 2h, obtain final product gene knockout plasmid.
Enzyme action connection procedure is as shown in Figure 1.
The preparation of 3.E.coli S17-1 competent cell
(1) activate E.coli, by the inoculum concentration of 1% (v/v), seed liquor be inoculated in 200mL SOB culture medium, 37 DEG C,
200rpm, cultivates to OD600For 0.6 about.
(2) culture is placed after 30min in frozen water, with the Mg of the pre-cooling of 15mL2+Solution, Ca2+Solution respectively washs bacterium
Body is once.
(3) use the resuspended thalline of 5mL glycerite, suspension bacteria liquid subpackage often pipe 40 μ L, be stored in -80 DEG C.
Competent cell preparation process is as shown in Figure 2.
4. gene knockout plasmid Transformed E .coli S17-1 competent cell
(1) take out after E.coli is placed in thawed on ice and add 10 μ L plasmids, gently mix, ice bath 30min.
It is immediately placed in 3min on ice after (2) 42 DEG C of thermal shock 90s, add the LB culture medium of 890 μ L, 30 DEG C, 200rpm trains
Foster 1h.
(3) 100 μ L conversional solution are taken to be coated on the LB flat board containing Kan, 30 DEG C of culture 18h.
Heat-shock transformed process is as shown in Figure 3.
5. engage conversion method and import plasmid to P.putida NBRC 14164
(1) respectively take E.coli the and P.putida bacterium solution 500 μ L after activation, be blended in same sterile centrifugation tube,
8000rpm is centrifuged 3min, be washed once with 1mL LB culture medium, adds the resuspended thalline of 1mL LB culture medium, 30 DEG C, 200rpm trains
Foster 12h, then stand 12h, first time homologous recombination completes.
(2) 100 μ L mixed liquors are coated on the flat board of LB-Amp-Kan, 30 DEG C of culture 24h.
(3) picking single bacterium colony, is inoculated in non-resistant LB fluid medium, 30 DEG C of culture 24h, and second homologous recombination is complete
Become.
(4) culture fluid is diluted 100 times with sterilized water, take 100 μ L to be coated on the flat board of LBS-Amp, 30 DEG C of cultures
24h.
(5) the bacterium picking single bacterium colony on LBS-Amp flat board, puts by number successively on LBS-Amp and LB-Kan flat board, and 30
DEG C culture 24h, mark growth had on LBS Amp flat board and on LB-Kan flat board the bacterium colony of no growth be positive bacterium colony.
(6) bacterium colony PCR, electrophoresis checking.
Twice homologous recombination process as shown in figure 4, before and after prs and rpiA gene knockout electrophoresis as shown in Figure 5.
Claims (7)
1. a kind of gene knockout method for pseudomonas putida NBRC 14164, is characterized in that step is as follows：
1) gene knockout plasmid construction；
2) preparation of E.coli S17-1 competent cell；
3) gene knockout plasmid Transformed E .coli S17-1 competent cell；
4) engage conversion method and import plasmid to P.putida NBRC 14164.
2. the method for claim 1, is characterized in that described step 1) be：Search and intend knocking out gene order, using soft
Primer is verified, two pairs of primer extension products differ 300- after part Primer Premier 5 design gene knockout primer and knockout
400bp, knocks out primer extension product and successfully constructs through enzyme action connection plasmid.
3. the method for claim 1, is characterized in that described step 2) be：For engaging the supplementary strain of conversion
E.coli prepares competent cell according to heat-shock transformed method, uses Mg2+Solution, Ca2+The washing of solution, whole sterile working.
4. the method for claim 1, is characterized in that described step 3) be：With heat-shock transformed method by gene knockout plasmid
Conversion enters E.coli competent cell.
5. the method for claim 1, is characterized in that described step 4) be：Homologous recombination will twice to engage trans-utilization
Gene knockout, first time homologous recombination plasmid enters P.putida from E.coli and is incorporated on chromosome, second homology
Recombiant plasmid is departed from from chromosome with the gene knocking out primer amplification, i.e. gene knockout success.
6. method as claimed in claim 5, is characterized in that：First time homologous recombination condition is for pseudomonas putida and containing weight
In 30 DEG C, 200rpm cultivates 12h to the escherichia coli of group suicide vector plasmid, then stands 12h.
7. method as claimed in claim 5, is characterized in that：Second homologous recombination condition is the first time homology weight after screening
Organize successful single bacterium colony to be inoculated in non-resistant LB fluid medium, 30 DEG C, 200rpm cultivates 24h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904228A (en) * | 2017-12-08 | 2018-04-13 | 中国水产科学研究院南海水产研究所 | A kind of method of the Vibrio harveyi homologous recombination gene knockout based on thermal shock |
| CN109182400A (en) * | 2018-08-09 | 2019-01-11 | 天津大学 | A method of improving Pseudomonas putida KT2440 acetic acid assimilative capacicy |
| CN110551749A (en) * | 2019-10-09 | 2019-12-10 | 武汉博欧特生物科技有限公司 | Construction method and application of pseudomonas putida suicide vector |
| CN112143689A (en) * | 2019-06-28 | 2020-12-29 | 中国科学院微生物研究所 | Construction of recombinant pseudomonas putida strain and application thereof in conversion of threonine to synthesize propionic acid |
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| CN103952429A (en) * | 2014-04-30 | 2014-07-30 | 上海交通大学 | Genetic engineering pseudomonas putida and construction method and application thereof |
| CN105296564A (en) * | 2015-10-29 | 2016-02-03 | 上海交通大学 | Method for preparing 3-succinyl pyridine by means of biotransformation |
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| CN102071185A (en) * | 2010-12-06 | 2011-05-25 | 南京师范大学 | Efficient gene knockout method of pseudomonas putida KT2440 |
| CN103952429A (en) * | 2014-04-30 | 2014-07-30 | 上海交通大学 | Genetic engineering pseudomonas putida and construction method and application thereof |
| CN105296564A (en) * | 2015-10-29 | 2016-02-03 | 上海交通大学 | Method for preparing 3-succinyl pyridine by means of biotransformation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107904228A (en) * | 2017-12-08 | 2018-04-13 | 中国水产科学研究院南海水产研究所 | A kind of method of the Vibrio harveyi homologous recombination gene knockout based on thermal shock |
| CN107904228B (en) * | 2017-12-08 | 2022-09-06 | 中国水产科学研究院南海水产研究所 | Vibrio harveyi homologous recombination gene knockout method based on heat shock |
| CN109182400A (en) * | 2018-08-09 | 2019-01-11 | 天津大学 | A method of improving Pseudomonas putida KT2440 acetic acid assimilative capacicy |
| CN112143689A (en) * | 2019-06-28 | 2020-12-29 | 中国科学院微生物研究所 | Construction of recombinant pseudomonas putida strain and application thereof in conversion of threonine to synthesize propionic acid |
| CN110551749A (en) * | 2019-10-09 | 2019-12-10 | 武汉博欧特生物科技有限公司 | Construction method and application of pseudomonas putida suicide vector |
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Application publication date: 20170308 |