CN106497958A - A kind of method of rapid build recombiant plasmid - Google Patents
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Abstract
The invention belongs to gene engineering technology field, discloses a kind of method of rapid build escherichia coli recombiant plasmid.Comprise the steps：The design of homologous recombination primer, carry pKD46 plasmids E.coli competent escherichia coli cells preparation, with cotransformation with the exogenous dna fragment of plasmid homology segment.Red recombinase is stably expressed using pKD46 plasmids under the induction of L arabinose, construct the coli strain containing Red recombination systems, recombinase can promote the section homologous with plasmid and linear plasmid entrained by exogenous dna fragment two ends to complete to recombinate, single restriction endonuclease sites that recombinant technique using RED exogenous dna fragment be inserted into carrier are achieved first, pKD46 plasmids can be eliminated under 37 DEG C of cultures, interference will not be produced to subsequent experimental, the construction step of recombiant plasmid is enormously simplify, the time is shortened.After the recombiant plasmid of generation is identified with enzyme action through PCR, positive colony efficiency is higher, can become a kind of new tool of construction recombination plasmid.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to a kind of side of rapid build escherichia coli recombiant plasmid
Method.
Background technology
In genetic engineering, one of most basic technology is construction recombination plasmid, needs genes of interest to be incorporated into vector plasmid.
The structure of conventional recombiant plasmid probably needs five steps：1. the acquisition of target DNA fragment, 2. carrier and target DNA fragment is double
Enzyme action, the 3. Ligation in vitro of carrier and target DNA double digestion fragment, 4. connection product be transformed into escherichia coli, 5. recon
Screening, generally requires 3-5 days, and restriction enzyme site dependence, loaded down with trivial details experimental implementation, relatively low recombination efficiency strongly limit function base
Because of a group development for research.
Along with the development of molecular biology, a kind of homologous recombination system based on bacteriophage lambda Red recombinases is applied to
Bacillus coli gene engineering research.By tri- genomic constitutions of bacteriophage lambda exo, bet, gam, they distinguish Red homologous recombination systems
Coding tri- kinds of protein of Exo, Beta, Gam.Exo albumen is a kind of exonuclease, and the molecular weight of single subunit is 24kD, Exo eggs
White activity form is a kind of cyclic trimer molecule, and one end that there is a hollow passage, passage centre can accommodate double-stranded DNA point
Son, the other end only accommodate single stranded DNA.Exo albumen may be incorporated in the end of double-stranded DNA, hold to 3 ' end drops from the 5 ' of DNA double chain
Solution DNA, makes DNA molecular form 3 ' sticky ends.Beta albumen plays decisive role in Red homologous recombination, with mediation
The function of complementary single-stranded dna annealing, is a kind of annealing albumen, and the molecular weight of single subunit is 25.8kD.In the solution, Beta albumen
Circulus is spontaneously formed, and is tightly combined the 3 ' jags in single stranded DNA, DNA is prevented by single-stranded nuclease degradation, and be situated between
Lead the annealing of complementary single-stranded dna.After the completion of double-stranded DNA annealing, Beta albumen is disintegrated down from DNA double chain.Gam albumen is
The peptide molecule of 16kD, can be combined with RecBCD exonucleases, suppress its Degradation to foreign DNA, prevent foreign DNA
Degraded by host after entering cell.In current Red homologous recombination systems, the exogenous DNA molecule with homology arm directly can lead to
Cross homology arm and find homology region, then carry out the restructuring of DNA.Red recombinant techniques are widely used in appoints to bacterial genomes
Meaning site carries out, in the genetic modification research of gene knockout or replacement, such as to make the linear target DNA fragments of importing using the technology
Homologous recombination is carried out with the particular sequence of genome, target gene is replaced by target gene so that the hereditary character of antibacterial is obtained
To change.
Content of the invention
In view of the loaded down with trivial details and recombinase vitro reactions of conventional construction of recombinant plasmid is expensive, the purpose of the present invention is to carry
For a kind of method quick, convenient, economic for the structure of recombiant plasmid.
The method that the present invention discloses recombiant plasmid in a kind of rapid build escherichia coli based on Red homologous recombination techniques,
It comprises the steps：
1) homologous recombination primer is designed：
(1) according to required plasmid vector, and genes of interest is inserted into the position of plasmid, chooses appropriate single limit
Property restriction enzyme site processed, does single endonuclease digestion with related restricted enzyme to carrier；
(2) two ends from linear carrier select the sequential design homologous sequence of appropriate size, unilateral homologous arm lengths
About 20-100bp；
(3) design PCR expands the primer of target DNA fragment, adds one side respectively with 5 ' ends of downstream primer at its upstream
Homology arm sequence；
2) competent escherichia coli cell for carrying pKD46 plasmids is prepared：
(1) pKD46 plasmids are transformed in escherichia coli, obtain the coli strain containing pKD46 plasmids；
(2) induce which to produce Red recombinases using L-arabinose, and be prepared into competent cell；
3) cotransformation：By with the exogenous dna fragment of plasmid homology segment and linear carrier by molecular number ratio 5:
1-10:Mix in the range of 1, be added to the competent escherichia coli cell for expressing Red recombinases, gently piping and druming is mixed, ice bath
20-30min, 42 DEG C of thermal shock 90s, is put in ice rapidly after thermal shock, ice bath 5-10min, adds appropriate LB or SOB to mix, 30 DEG C
Shaking table recovery 45-60min, after centrifugation, is applied on the solid LB flat boards of corresponding resistant, 37 DEG C of overnight incubations；
4) recombiant plasmid is extracted；
5) identify:The recombiant plasmid for extracting is entered performing PCR identification, enzyme action identification；
6) bacterial strain containing positive recombinant plasmid is protected bacterium, -70 DEG C frozen.
The principle schematic of exogenous dna fragment construction recombination plasmid such as Fig. 1 used in RED recombination systems.
Preferably, the escherichia coli of described carrying pKD46 plasmids are BJ5183, XL2-Blue, DH5 α.
The present invention using pKD46 plasmids L-arabinose induction under stably express Red recombinases, construct containing
The coli strain of Red recombination systems, recombinase can promote the section homologous with plasmid entrained by exogenous dna fragment two ends
Complete to recombinate with linear plasmid, achieve the single limit that exogenous dna fragment is inserted into the recombinant technique using RED carrier first
Property restriction enzyme site processed, can eliminate the plasmid under 37 DEG C of cultures, will not produce interference to subsequent experimental.Enormously simplify weight
The construction step of group plasmid, shortens the time, and this process is only needed one day, and traditional method needs 2-3 days.The recombiant plasmid warp of generation
After PCR is identified with enzyme action, positive colony efficiency is higher, can become a kind of new tool of construction recombination plasmid.
Description of the drawings
Fig. 1 is the principle schematic of exogenous dna fragment construction recombination plasmid used in RED recombination systems.
Fig. 2 is the electrophoretogram of 1 recombiant plasmid PCR of experimental group identifications
Purpose band size about 810bp.M is DL2000；1 is positive control；2nd, 3,4,5,6 is recombiant plasmid；7 are feminine gender
Control.
Fig. 3 is the electrophoretogram of 2 recombiant plasmid PCR of experimental group identifications
Purpose band size about 800bp.M is DL2000；1 is positive control；2nd, 3,4,5,6 is recombiant plasmid；7 are feminine gender
Control.
Fig. 4 is the electrophoretogram of 1 recombiant plasmid Kpn I＆Xho I enzyme action of experimental group identification
Purpose band size about 770bp, carrier size about 5880bp.M is DL2000；1 produces for pET-41a plasmid enzyme restrictions
Thing；2nd, 3,4,5,6 are restructuring plasmid enzyme restriction product.
Fig. 5 is the electrophoretogram of 2 recombiant plasmid Kpn I＆Xho I enzyme action of experimental group identification
Purpose band size about 770bp, carrier size about 5880bp.M is DL2000；1 produces for pET-41a plasmid enzyme restrictions
Thing；2nd, 3,4,5,6 are restructuring plasmid enzyme restriction product.
Fig. 6 is the electrophoretogram of 1 recombiant plasmid BamH I＆Hind III digestions of experimental group identification
Purpose band size about 840bp, carrier size about 5810bp.M is DL2000；6 produce for pET-41a plasmid enzyme restrictions
Thing；1st, 2,3,4,5 are restructuring plasmid enzyme restriction product.
Fig. 7 is the electrophoretogram of 2 recombiant plasmid BamH I＆Hind III digestions of experimental group identification
Purpose band size about 840bp, carrier size about 5810bp.M is DL2000；1 produces for pET-41a plasmid enzyme restrictions
Thing；2nd, 3,4,5,6 are restructuring plasmid enzyme restriction product.
Specific embodiment
Embodiment 1
First, the design of homologous recombination primer
(1) with enhanced green fluorescence protein (EGFP) gene as design object；PET-41a is recombinant vector, is used
EcoR I linearisations, the unilateral homologous section length that designs are that the forward primer of 45bp is：
Downstream primer is：
Unilateral homologous section length is that the forward primer of 35bp is：
Downstream sequence is：
Italic underscore part is EcoR I sites two ends (including EcoR I restriction enzyme site sequences) on pET-41a carriers
Sequence (for carrying out homologous recombination with linear pET-41a), remaining sequence for amplification EGFP gene.
2nd, the preparation of the BJ5183 competence antibacterial of pKD46 plasmid is carried
(1) the BJ5183 bacterial strains single bacterium colony inoculation of the carrying pKD46 plasmids obtained from flat board picking General transformation method
Contain in the SOB culture medium of Amp (final concentration 25-50 μ g/mL) to 4mL, 30 DEG C, 220rpm, shaken cultivation is overnight.
(2) take 1mL (inoculum concentration 1%～2%) culture be inoculated into dress 100mL contain Amp (final concentration 25-50 μ g/mL)
In the 250mL conical flasks of SOB culture medium, 30 DEG C, 220rpm, first shaken cultivation 1h are subsequently adding L-arabinose (final concentration
0.2%) inducing culture is to OD600=0.4～0.5.
(3) cultured bacterium solution is transferred in 2 sterilized 50mL centrifuge tubes in sterilized super-clean bench, ice bath
30min.
(4) 2 DEG C, 1000g centrifugation 5min.Abandoning supernatant in super-clean bench, being upside down on filter paper makes liquid flow as far as possible
To the greatest extent.
(5) first respectively with the CaCl of the cold 0.1mol/L of 2mL2Solution suspension thalline (gently patted mixed upward with handss by thalline
Even to bacterium solution be in cloud), then plus CaCl2Solution is mixed to 30mL, ice bath 20min.
(6) 2 DEG C, 1000g centrifugation 5min.Abandoning supernatant in super-clean bench, being upside down on filter paper makes liquid flow as far as possible
To the greatest extent.
(7) repeat step (4), (5) and (6) three times.
(8) first respectively with the glycerol CaCl that 0.5mL is cold2Solution (0.1mol/L CaCl2+ 15% glycerol) suspension thalline (bacterium
It is in cloud that body gently pats mixing to bacterium solution with handss upward), then glycerol adding CaCl2Solution is mixed to 2mL, and subpackage is to sterilizing
1.5mL EP pipes in (often 100 μ L of pipe).Use is preserved under the conditions of -70 DEG C for a long time can.
3rd, the acquisition containing EGFP gene recombination sequence
With pEGFP-N1 as template；Upstream and downstream primer used by PCR has two groups, and it is 45bp that one group is unilateral homologous section length
Primer, another group be unilateral homologous section length be 35bp primer；Premix LA of the enzyme used by PCR for TaKaRa companies
Taq Version 2.0.
(1) in sterilized microcentrifugal tube, successively plus following component：Premix LA Taq 12.5μL、Sense
primer(10μM)1μ、Anti-sense primer(10μM)1μL、pEGFP-N1(100ng/μL)1μL、ddH2O 9.5μL.
After mixing, first round amplification is carried out in PCR instrument.Response procedures：PCR cycle condition is as follows：94 DEG C of denaturations
5min, 94 DEG C of degeneration 30s, anneal 58 DEG C of 30s, extends 72 DEG C of 1min, 35 circulations, 72 DEG C of extension 10min.
(2) PCR primer is reclaimed with DNA QIAquick Gel Extraction Kits, obtain target DNA fragment.
4th, the linearisation of pET-41a carriers
(1) EcoR I single endonuclease digestion pET-41a carriers are used, in sterilized microcentrifugal tube, successively plus following component：
pET-41a 5-10μg、10×H Buffer 5μL、EcoRI(15U/μL)0.5μL、ddH2The μ L of O to 50.
(2) 37 DEG C of water-baths 4 hours.
(3), after enzyme action terminates, electrophoresis is carried out with plain agar sugar gel, then cut target stripe and returned using DNA gel
Receive test kit and reclaim digestion products.
5th, linearisation pET-41a and target DNA fragment cotransformation
(1) experimental group 1 is 45bp target DNAs for taking 200ng linearisation pET-41a carriers and 200-300ng homology segments
The mixture of fragment；Experimental group 2 is 35bp purposes for taking 200ng linearisation pET-41a carriers and 200-300ng homology segments
The mixture of DNA fragmentation；Matched group is converted in the table of the above-mentioned preparations of 100 μ L respectively for taking 200ng linearisation pET-41a carriers
Reach in the BJ5183 competence of Red recombinases.Ice bath 30min.
(2) 42 DEG C of insulation 90s carry out thermal shock.It is put in ice rapidly after thermal shock, ice bath 10min.
(3) after adding 900 μ L SOB culture medium to mix, 30 DEG C of vibration (shaking speed is less than 150rpm) culture 60min,
Antibacterial is made to restore normal growth state, and the antibiotic resistance gene of expression plasmid coding.
(4) 5000rpm centrifugations 3min under room temperature.800 μ L of supernatant liquid are carefully sucked with liquid-transfering gun.
(5) again will be whole on the flat board of the LB solid mediums containing Kan (25 μ g/mL of final concentration) after the mixing of remaining bacterium solution
Coating, stands 1～2min.Overnight incubation (will typically cultivate 16h) is inverted in 37 DEG C of incubator, pKD46 matter can be removed
Grain.
(6) culture is completed, and matched group place flat board is not long, and experimental group place flat board grows single bacterium colony, chooses list from flat board
Bacterium colony carries plasmid identification.
6th, the extraction of recombiant plasmid
(1) on random picking flat board, several single bacterium colonies are inoculated into LB liquid trainings of the 5mL containing Kan (25 μ g/mL of final concentration)
In foster base, 37 DEG C of shaken cultivation are overnight.
(2) will be shaken to mixed turbid bacterium solution carries out guarantor bacterium in sterilized super-clean bench first, takes 600 μ L bacterium solutions in sterilized
Sterilized 50% glycerol of 400 μ L is added in 1.5mL EP pipes, -70 DEG C of refrigerators are frozen, the commonly little upgrading grain examination of remaining bacterium solution
Agent box carries out plasmid extraction.
7th, PCR identifications
(1) template is done as positive control with pEGFP-N1, template is done as negative control with ddH2O, primer amplification purpose
The recombiant plasmid for extracting is entered performing PCR identification by the respective primer of gene.
To in sterilized microcentrifugal tube, successively plus following component：dNTP(2.5mM each)2μL、Sense
primer(10μM)0.5μL、Anti-sense primer(10μM)0.5μL、10×PCR Buffer 2.5μL、rTaq(5U/μ
L) 0.2 μ L, template 1 μ L, ddH2O 18.3μL.After mixing, first round amplification is carried out in PCR instrument.Response procedures：PCR cycle
Condition is as follows：94 DEG C of denaturations 5min, 94 DEG C of degeneration 30s, anneal 58 DEG C of 30s, 72 DEG C of 1min of extension, 30 circulations, and 72 DEG C are prolonged
Stretch 10min.(2) PCR primer is identified (such as Fig. 2 and Fig. 3 by plain agar sugar gel electrophoresiss.Fig. 2 is experimental group 1, and Fig. 3 is real
Test group 2.).
8th, enzyme action identification
(1) positive recombinant vector that PCR is identified is transformed into upgrading grain in bacillus coli DH 5 alpha and does enzyme action identification.
(2) restriction enzyme that is identified respectively as double digestion using Kpn I＆Xho I and BamH I＆Hind III
Enzyme, the former have on that is, linear pET-41a and target DNA fragment in designed recombination sequence, and the latter is in recombination sequence
Outward, i.e., have on only linear pET-41a.With pET-41a as negative control, the recombiant plasmid for extracting is carried out enzyme action identification.
(3) to sterilized PCR pipe 1. in, sequentially add following component：Recombiant plasmid 1-3 μ g, 10 × M Buffer
2μL、Kpn I(10U/μL)0.2μL、Xho I(10U/μL)0.2μL、ddH2The μ L of O to 20.To sterilized PCR pipe 2. in,
Sequentially add following component：Recombiant plasmid 1-3 μ g, 10 × K Buffer, 2 μ L, BamH I (15/ μ L) 0.2 μ L, Hind III
(15U/μL)0.2μL、ddH2The μ L of O to 20.
(4) 37 DEG C of water-baths 2 hours.After enzyme action terminates, electrophoresis is carried out with plain agar sugar gel (if Fig. 4 and Fig. 5 is Kpn
I＆Xho I double digestion results, Fig. 4 are experimental group 1, and Fig. 5 is experimental group 2.Fig. 6 and Fig. 7 is tied for BamH I＆Hind III double digestions
Really, Fig. 6 is experimental group 1, and Fig. 7 is experimental group 2.).
(5) bacterial strain containing positive recombinant plasmid is protected bacterium, -70 DEG C frozen.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, other any spirit without departing from the present invention and the change, modification, replacement that is made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
SEQUENCE LISTING
<110>Wuhan Bioengineering Institute
<120>A kind of method of rapid build recombiant plasmid
<160> 4
<170> PatentIn version 3.5
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<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> APM1FA(sense)
<400> 1
ccggtgatga cgacgacaag agtcccatgg gatatcgggg atccgaatta tggtgagcaa 60
gggcgag 67
<210> 2
<211> 67
<212> DNA
<213> Artificial Sequence
<220>
<223> APM1FS(antisense)
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gcaagcttgt cgacggagct cgcctgcagg cgcgccaagg cctgtacagt tacttgtaca 60
gctcgtc 67
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<212> DNA
<213> Artificial Sequence
<220>
<223> BRA2FS(sense)
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cgacgacaag agtcccatgg gatatcgggg atccgaatta tggtgagcaa gggcgag 57
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<212> DNA
<213> Artificial Sequence
<220>
<223> BRA2FA(antisense)
<400> 4
cgacggagct cgcctgcagg cgcgccaagg cctgtacagt tacttgtaca gctcgtc 57
Claims (1)
1. in a kind of rapid build escherichia coli based on Red homologous recombination techniques recombiant plasmid method, it is characterised in that bag
Include following steps：
1）Design homologous recombination primer：
（1）According to required plasmid vector, and genes of interest is inserted into the position of plasmid, chooses appropriate single restricted
Restriction enzyme site, does single endonuclease digestion with related restricted enzyme to carrier；
（2）Two ends from linear carrier select the sequential design homologous sequence of appropriate size, unilateral homologous arm lengths about 20-
100bp；
（3）Design PCR expands the primer of target DNA fragment, at its upstream with the 5 ' of downstream primer ends respectively plus unilateral homologous
Arm sequence；
2）Prepare the competent escherichia coli cell for carrying pKD46 plasmids：
（1）PKD46 plasmids are transformed in escherichia coli, the coli strain containing pKD46 plasmids is obtained；
（2）Induce which to produce Red recombinases using L-arabinose, and be prepared into competent cell；
3）Cotransformation：By with the exogenous dna fragment of plasmid homology segment and linear carrier by molecular number ratio 5:1-10:
Mix in the range of 1, be added to the competent escherichia coli cell for expressing Red recombinases, gently piping and druming is mixed, ice bath 20-
30min, 42 DEG C of thermal shock 90s, is put in ice rapidly after thermal shock, ice bath 5-10min, adds appropriate LB or SOB to mix, 30 DEG C of shaking tables
Recovery 45-60min, after centrifugation, is applied on the solid LB flat boards of corresponding resistant, 37 DEG C of overnight incubations；
4）Extract recombiant plasmid；
5）Identification:The recombiant plasmid for extracting is entered performing PCR identification, enzyme action identification；
6）Bacterial strain containing positive recombinant plasmid is protected bacterium, -70 DEG C frozen.
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| CN201611023368.5A CN106497958A (en) | 2016-11-21 | 2016-11-21 | A kind of method of rapid build recombiant plasmid |
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Cited By (3)
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| CN108841866A (en) * | 2018-06-04 | 2018-11-20 | 云舟生物科技（广州）有限公司 | A kind of adenovirus vector and its construction method |
| CN109735565A (en) * | 2018-12-29 | 2019-05-10 | 浙江大学 | The method and purposes of tracer pig GADD45a subcellular localization |
| CN111004813A (en) * | 2019-12-27 | 2020-04-14 | 苏州泓迅生物科技股份有限公司 | Super-large plasmid construction kit, super-large plasmid construction method and application thereof |
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| CN101899467A (en) * | 2009-05-26 | 2010-12-01 | 上海捷瑞生物工程有限公司 | Method for inserting target DNA fragment into vector |
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| CN1162546C (en) * | 2002-07-30 | 2004-08-18 | 复旦大学 | Method of constructing genetic engineering organism based on in vivo isogenesis recombination |
| CN102124112A (en) * | 2008-09-10 | 2011-07-13 | 金斯瑞公司 | Homologous recombination-based DNA cloning methods and compositions |
| CN101899467A (en) * | 2009-05-26 | 2010-12-01 | 上海捷瑞生物工程有限公司 | Method for inserting target DNA fragment into vector |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841866A (en) * | 2018-06-04 | 2018-11-20 | 云舟生物科技（广州）有限公司 | A kind of adenovirus vector and its construction method |
| CN108841866B (en) * | 2018-06-04 | 2022-07-05 | 云舟生物科技(广州)股份有限公司 | Adenovirus vector and construction method thereof |
| CN109735565A (en) * | 2018-12-29 | 2019-05-10 | 浙江大学 | The method and purposes of tracer pig GADD45a subcellular localization |
| CN111004813A (en) * | 2019-12-27 | 2020-04-14 | 苏州泓迅生物科技股份有限公司 | Super-large plasmid construction kit, super-large plasmid construction method and application thereof |
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