CN110066788A - A kind of gene with multiple DNA fragments high efficiency assemble method of zero background - Google Patents
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Abstract
The invention discloses a kind of gene with multiple DNA fragments high efficiency assemble method of zero background, include the following steps: to construct puc57-CATP carrier；Building clone has the plasmid of CATP genetic fragment；Selection target gene is divided into small fragment according to mrna length and carries out gene chemical synthesis, and gene both ends add two fermentoid sites, are connected to cloning vector；Multiple small fragments include that the CATP genetic fragment constructed is assembled with Golden gate cloning reaction, use T7DNA polymerase as ligase, assemble product and convert Escherichia coli, the LB plate for being coated on ammonia benzyl and chlorampenicol resistant is screened.Vector plasmid is designed by unique carrier and segment without first passing through the processing such as linearisation in advance, disposably while efficiently assembles multiple genetic fragments；Using the higher T7DNA ligase of fidelity, the probability that segment correctly assembles is improved.
Description
Technical field
The present invention relates to field of biotechnology, the gene with multiple DNA fragments high efficiency assemble method of specifically a kind of zero background.
Background technique
Gene cloning is acquired a great achievement in terms of agricultural, animal husbandry and medicine in recent years, the basic principle is that external source
Gene and cloning vector carry out Ligation in vitro, then convert host cell, filter out the conversion containing target gene recombinant plasmid
Son.
Traditional gene cloning step includes: selection target gene and designs corresponding primer；Purpose base is expanded with primer PCR
Because of segment；The cloning vector (for fidelity amplification) of suitable (resistance marker, restriction enzyme site etc.) is selected, and PCR fragment is connected
Enter in cloning vector, (generally can carry an A at end with the PCR product of Taq enzyme, can Solution 1 effect under with
The linear carrier of both ends one T of each band connects).
With the development of molecular biology, people increase the synthesis demand of polygenes segment.Golden gate gram
Grand is one of common method, and principle is to add IIs type restriction enzyme enzyme recognition site at small fragment both ends, and utilization is same
The different cohesive ends that kind restriction enzyme generates, to disposably assemble multiple genetic fragments.
However the T4 DNA ligase that conventional Golden gate cloning process uses can be catalyzed cohesive end or flat end
It is combined between end double-stranded DNA or the 5 ' ends-P and the end 3 '-OH of RNA with phosphodiester bond, meets 3/4 consistent two viscosity
End can be annealed by catalysis, and secondly the fidelity of T4 DNA ligase is lower, therefore be existed using traditional T4 DNA ligase
Need carefully to design the cohesive ends of multiple segments when being Golden gate clone, to avoid occur cohesive end 3/4 or
Situation as reversed 3/4 occurs, and designs unreasonable Golden gate clone, is often difficult to using T4 DNA ligase
It is correctly cloned, connection is tended to form less segment interconnection, so that joint efficiency further decreases.
Traditional Golden gate clone products after converting Escherichia coli, still suffer from a small amount of ligase by carrier and
The small fragment mistake that carrier is digested connects or connects less, and does not cut clean carrier and a small amount of pcr template, in this way for subsequent
Bacterium colony screening forms very big background interference.These following background interferences can during Escherichia coli are replicated by
It is gradually enriched with, occupies the overwhelming majority of plate clone total number, assembling success rate is caused to be greatly lowered, or even assembling failure.
Therefore, we invent and improve a kind of method for the high efficiency recombination of zero background in cloning procedure.
Vector plasmid can carry out digestion with restriction enzyme without first passing through the processing such as linearisation in advance in same system
And connection, effectively improve the accuracy of multiple clips connection；And it, can by artificial additionally one section of chloramphenicol resistance gene of addition
The jamming pattern for avoiding cloning vector itself and pcr template from generating, carries out the seamless connection of up to 10 segments.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of efficient polygenes segment buildings of zero background
Method, to solve the problems, such as to propose in background technique.
A kind of cloning process of the efficient assembling gene of zero background, step include:
Step 1 constructs puc57-CATP carrier；
Step 2 building clone has the plasmid of CATP genetic fragment；
Step 3 selection target gene is divided into small fragment according to mrna length and carries out gene chemical synthesis, gene both ends addition two
Fermentoid site, is connected to cloning vector；
The multiple small fragments of step 4 include that the CATP genetic fragment constructed carries out group with Golden gate cloning reaction
Dress, uses T7 archaeal dna polymerase rather than T4 archaeal dna polymerase is as ligase, can be improved the probability correctly assembled.Assembling produces
Object converts Escherichia coli, and the LB plate for being coated on ammonia benzyl and chlorampenicol resistant is screened.
Preferably, step 1 is specifically divided into following steps:
(1) puc57-CATP carrier includes replicon, ampicillin resistance and multiple cloning sites region, with pCATP-PF/
PCATP-PR is expanded respectively as upstream and downstream primer PCR obtains product, and recovery purifying.
(2) using puc57 as carrier, double digestion is carried out with EcoR I and HindIII, using column recovery purifying digestion products,
Linearized vector after PCR product and digestion that step (1) is obtained is recombinated with Gibson to be connected.
(3) recombinant products convert Escherichia coli TOP10 competent cell, the specific steps are as follows:
A. the TOP10 competent cell and 5 μ L connection products that 50 μ L melt on ice are added in 2mL centrifuge tube, gently mixes
It is even.
B. after centrifuge tube being placed 30min on ice, centrifuge tube is placed on ice by the heat shock 60s in 42 DEG C of water-baths immediately
Cooling 2min.
C. 600 μ L LB liquid mediums are added, mix gently, 37 DEG C, 200rpm, constant-temperature shaking culture 45min.
D.5000rpm it is centrifuged 5min, stays 200 μ L bacterium solutions to be applied on the LB solid medium of IPTG and X-gal, wherein solid
The culture medium benzyl mycin of ammonia containing 50mg/L；
E. coated plate is just being put in 5min in 37 DEG C of insulating boxs, then is inverted culture 12h and grows monoclonal.
(4) identification of recombinant clone
The multiple monoclonals of picking are in 4mL LB liquid medium, wherein fluid nutrient medium ammonia containing 50mg/L benzyl mycin, and 37
DEG C shaken cultivation 12h is simultaneously sequenced to bacterium solution muddiness, extracting plasmid.
Preferably, step 2 is specifically divided into following steps:
(1) CATP plasmid is the reading frame sequence of chloramphenicol resistance gene, does not include initiation codon ATG and upstream
Promoter sequence, gene both ends are two fermentoid restriction enzyme sites (see " Fig. 3 "), the segment viscosity end formed after two fermentoid BsaI digestions
End is respectively GCTT and ACGG, and cohesive end ACGG is connected with the carrier puc57-CATP after BsaI digestion.
(2) using pACYC-Duet1 as template, with primer CATP-PF/CATP-PR amplified fragments, obtained PCR fragment glue
The pUC57 carrier of flush end connection EcoRV linearisation after recycling.
(3) connection product converts Escherichia coli TOP10 competent cell
It is identical as this part of step 1.
(4) identification of recombinant clone
It is identical as this part of step 1.
Preferably, step 3 is specifically divided into following steps:
(1) target fragment is divided into small fragment, and adds two fermentoid sites
Target gene is divided into small fragment according to length without two fermentoid site BsaI, target gene, designs head and the tail primer amplification
Small fragment, or the small fragment by gene chemical synthesis synthesis target gene.Add two sites fermentoid BsaI and viscosity end in gene both ends
End, the cohesive end design principle of multiple genetic fragments are as follows: first 5 ' cohesive end of segment is consistent with linearized vector head,
For GAAT；3 ' cohesive ends of the last one segment are consistent with linearized vector tail portion, are GCTT；The 4 of different genes segment
The cohesive end of bp is different, mutually not reverse complemental, avoids the occurrence of in 4 bases that there are continuous 3 bases are identical or reversed
It is complementary.
(2) small fragment is connected to cloning vector
After the PCR product glue recycling of several small fragments, it is connected respectively to the pUC57 carrier of EcoRV linearisation.
(3) connection product converts Escherichia coli TOP10 competent cell
It is identical as this part of step 1.
(4) identification of recombinant clone
It is identical as this part of step 1.
Preferably, step 4 is specifically divided into following steps:
(1) according to following system in 0.2mL PCR pipe configuration group reaction cartridge.
Preparation is performed as follows in reaction system:
(2) after the completion of reaction system configuration, 0.2mL PCR pipe is placed in PCR instrument and covers rubber pad, according to following
Response procedures are arranged in PCR reaction condition.Pay attention to that the lid of PCR instrument is had to cover tightly.
It is as follows that PCR instrument response procedures are set:
(3) connection product converts
A. the TOP10 competent cell and the 5 above-mentioned recombinant products of μ L that 50 μ L melt on ice are added in 2mL centrifuge tube, gently
It is light to mix.
B. after centrifuge tube being placed 30min on ice, centrifuge tube is placed on ice by the heat shock 60s in 42 DEG C of water-baths immediately
Cooling 2min.
C. 600 μ L LB liquid mediums are added, mix gently, 37 DEG C, 200rpm, constant-temperature shaking culture 45min.
D.5000rpm it is centrifuged 5min, stays 200 μ L bacterium solutions to be applied on LB solid medium, wherein solid medium contains
50mg/L ammonia benzyl mycin and 12.5mg/L chloramphenicol；
E. coated plate is just being put in 5min in 37 DEG C of insulating boxs, then is inverted culture 12h and grows monoclonal.
(4) identification of recombinant clone
It is identical as this part of step 1.
The beneficial effects of the present invention are:
The gene with multiple DNA fragments high efficiency assemble method of zero background of the invention is mainly cut using II type restriction enzyme
Site is located at the characteristics of on the outside of recognition site, and design introduces different target fragments with the PCR primer of specified viscosity end.Before
Phase T7 DNA ligase improves the probability that segment correctly assembles, and later period ammonia benzyl and chloramphenicol double resistance screen are recombinated
Accuracy is up to 100% positive colony.The program has the advantages that
1) present invention includes the applicable carrier of building: puc57-CATP carrier, CATP genetic fragment plasmid；
2) cloning vector and expression vector plasmid are not necessarily to first pass through the processing such as linearisation in advance, reduce cost；
3) unique carrier and segment design, and the higher T7DNA ligase of fidelity is used, it is disposable while efficient
Correctly assemble multiple genetic fragments；
4) by utilizing Double-resistant artificially in CATP (chloramphenicol) gene promoter one section of CATP gene of downstream connection
(the ammonia benzyl resistance that carrier carries and the chloromycetin gene additionally introduced) screening avoids original clone carrier and original original segments
The interference of carrier reasons for its use；
5) seamless connection of up to 10 segments of successful clone.
Vector plasmid is designed, one without first passing through the processing such as linearisation in advance by unique carrier and segment
Secondary property efficiently assembles multiple genetic fragments simultaneously；Using the higher T7 DNA ligase of fidelity, improves segment and correctly assemble
Probability；After recombinant products convert Escherichia coli, it is coated on ammonia benzyl and what the Double LB plate screening of chloramphenicol obtained is
Positive colony avoids empty carrier background interference；In successful clone up to 10 segments to destination carrier puc57-CATP, confrontation
Grain carries out digestion and sequence verification, the results showed that clone's positive rate is up to 100%.
Detailed description of the invention
Fig. 1 is puc57-CATP plasmid map.
Fig. 2 is the schematic diagram that the segment with different cohesive ends connects puc57-CATP.
Growth of the Fig. 3 for linear carrier and the recombinant bacterium of 10 segments connection on different ligases and different resistant panels
Figure.
Fig. 4 is the recombinant monoclonal bacterium bacterium solution detection grown on different ligases and different resistant panels.
Specific embodiment
Below by way of specific embodiment, the present invention is further elaborated.
1. strain
Host strain Escherichia coli TOP10.
2. reagent
Archaeal dna polymerase and dNTP are general biological (system) Co., Ltd in Anhui；
Glue recycling and the small pumping kit of plasmid are AXYGEN product；
T4 DNA ligase is purchased from Thermo company；
T7 DNA ligase, restriction enzyme Bsa I, EcoR I, HindIII are purchased from NEB company；
Primer synthesizes our company's synthesis, and pUC57 and pACYC-duet are the commercial carrier that our company saves；Recombinase
GenREC is our company's development & production.
1. experiment flow
A kind of cloning process of the efficient assembling gene of zero background, step include:
(1) puc57-CATP carrier is constructed；
(2) building clone has the plasmid of CATP genetic fragment；
(3) selection target gene is divided into small fragment according to mrna length and carries out gene chemical synthesis, and two fermentoids are added at gene both ends
Site is connected to cloning vector；
(4) multiple small fragments include that the CATP genetic fragment constructed is assembled with golden gate cloning reaction, are made
It uses T7DNA polymerase rather than T4DNA polymerase is as ligase, can be improved the probability correctly assembled.Assemble product conversion
Escherichia coli, the LB plate for being coated on ammonia benzyl and chlorampenicol resistant are screened.
Specific experiment process is as follows:
Product, gel extraction PCR product pCATP- are obtained with two primer Overlap extension PCRs of pCATP-PF/pCATP-PR
V.Using pACYC-Duet1 as template, with primer CATP-PF/CATP-PR amplified fragments, gel extraction obtains PCR fragment CATP.
Nine genes are respectively designated as RecA1, RecA2, RecA3, RecA4, RecA5, RecA 6, RecA 7, RecA
8,9 RecA, each small fragment size are about 100-110bp, and size is about 1000bp after assembling.Nine genes are sequentially connected
Cohesive end is respectively gaat-ggaa-taca-gaac-aggc-gtat-aacg-tgac-ccgc-gctt, from e. coli k12
Nine target gene of genome amplification.
Its upstream and downstream primer title is shown in Table lattice one.
Table one
Using puc57 as carrier, double digestion is carried out with EcoR I and Hind III, uses column recovery purifying digestion products.With
Puc57 is carrier, carries out digestion with EcoR V, uses column recovery purifying digestion products.
By the puc57 GenRec recombinase weight of PCR fragment pCATP-V and EcoR I and the Hind III with linearisation
Preparation is performed as follows in group, reaction system:
Recombination method is as follows: after above-mentioned raw materials are mixed, recombining reaction is carried out in 50 DEG C of water-bath, the reaction time is
15 minutes.2 μ L are taken to convert Escherichia coli Top10 competent cell after reaction.
By CATP gene, mini gene segment the being separately connected property puc57 carrier of nine RECA, reaction system by as follows into
Row is prepared:
Connection procedure, specifically includes the following steps: being connected 30 minutes after above-mentioned raw materials are mixed in 22 DEG C of room temperatures.
Connection product is converted into Escherichia coli Top10 competent cell:
A. it takes the 5 above-mentioned recombinant products of μ L to be transferred in 50 μ L TOP10 competent cells, mixes gently.
B. after centrifuge tube being placed 30min on ice, centrifuge tube is placed on ice by the heat shock 60s in 42 DEG C of water-baths immediately
Cooling 2min.
C. 600 μ L LB liquid mediums are added, mix gently, 37 DEG C, 200rpm, constant-temperature shaking culture 45min.
D.5000rpm it is centrifuged 5min, stays 200 μ L bacterium solutions to be applied on LB solid medium, wherein solid medium contains
50mg/L ammonia benzyl mycin；
E. coated plate is just being put in 5min in 37 DEG C of insulating boxs, then is inverted culture 12h and grows monoclonal.
Next day grows to plate clone, and the plate of 9 different genes 3 monoclonals of equal picking are in 4mL LB Liquid Culture
In base, wherein fluid nutrient medium ammonia containing 50mg/L benzyl mycin, 37 DEG C of 220rpm shaken cultivation 12h extract plasmid to bacterium solution muddiness
And it is sequenced.
The plasmid order-checking of small fragment gene correctly enters in next step afterwards, i.e., multiple clips assembles.
The schematic diagram of segment connection puc57-CATP with different cohesive ends is as shown in Figure 2.
Multiple genetic fragment assemblings configuration group reaction cartridge in 0.2mL PCR pipe according to following system:
After the completion of reaction system configuration, following procedure is set in PCR instrument,
(3) connection product converts
A. the TOP10 competent cell and the 5 above-mentioned recombinant products of μ L that 50 μ L melt on ice are added in 2mL centrifuge tube, gently
It is light to mix.
B. after centrifuge tube being placed 30min on ice, centrifuge tube is placed on ice by the heat shock 60s in 42 DEG C of water-baths immediately
Cooling 2min.
C. 600 μ L LB liquid mediums are added, mix gently, 37 DEG C, 200rpm, constant-temperature shaking culture 45min.
D.5000rpm be centrifuged 5min, stay 200 μ L bacterium solutions be applied on LB solid medium (the benzyl mycin of ammonia containing 50mg/L and
12.5mg/L chloramphenicol).
E. coated plate is just being put in 5min in 37 DEG C of insulating boxs, then is inverted culture 12h and grows monoclonal.
(4) identification of recombinant clone
3 monoclonals of picking are in 4mL LB liquid medium, wherein fluid nutrient medium ammonia containing 50mg/L benzyl mycin, and 37 DEG C
Shaken cultivation 12h is muddy to bacterium solution, and bacterium colony PCR identifies positive colony.
Multiple clips is binned in the lithograph of different ligases and different resistances, as shown in Figure 3.
Multiple clips is binned in bacterium colony PCR identification positive colony figure after different ligases and the growth of different resistant panels, such as Fig. 3
It is shown.
Multiple clips is binned in positive clone rate statistical form after different ligases and the growth of different resistant panels, such as two institute of table
Show.
Table two
The multiple cloning sites sequence of carrier puc57-CATP
Note:WithIt is the cleavage site of BsaI；Gagacc and ggtctc is the recognition site of BsaI；Ash
Color dash area is chloromycetin gene promoter.
CATP fragment sequence:
Note:WithIt is the cleavage site of BsaI；Gagacc and ggtctc is the recognition site of BsaI
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (5)
1. a kind of gene with multiple DNA fragments high efficiency assemble method of zero background, which comprises the steps of:
Step 1 constructs puc57-CATP carrier；
Step 2 building clone has the plasmid of CATP genetic fragment；
Step 3 selection target gene is divided into small fragment according to mrna length and carries out gene chemical synthesis, and two fermentoids are added at gene both ends
Site is connected to cloning vector；
The multiple small fragments of step 4 include that the CATP genetic fragment constructed is assembled with Golden gate cloning reaction, are made
It uses T7 archaeal dna polymerase as ligase, assembles product and convert Escherichia coli, be coated on the LB plate of ammonia benzyl and chlorampenicol resistant
It is screened.
2. the gene with multiple DNA fragments high efficiency assemble method of zero background according to claim 1, it is characterised in that: step 1 tool
Body is divided into following steps:
(1) puc57-CATP carrier includes replicon, ampicillin resistance and multiple cloning sites region, with pCATP-PF/
PCATP-PR is expanded respectively as upstream and downstream primer PCR obtains product, and recovery purifying；
(2) using puc57 as carrier, double digestion is carried out with EcoR I and HindIII, using column recovery purifying digestion products, will be walked
Suddenly the linearized vector after (1) obtains PCR product and digestion is recombinated with Gibson to be connected；
(3) recombinant products convert Escherichia coli TOP10 competent cell, the specific steps are as follows:
A. the TOP10 competent cell and 5 μ L connection products that 50 μ L melt on ice are added in 2mL centrifuge tube, mixes gently；
B. after centrifuge tube being placed 30min on ice, centrifuge tube is placed in cooled on ice immediately by the heat shock 60s in 42 DEG C of water-baths
2min；
C. 600 μ L LB liquid mediums are added, mix gently, 37 DEG C, 200rpm, constant-temperature shaking culture 45min；
D.5000rpm it is centrifuged 5min, stays 200 μ L bacterium solutions to be applied on the LB solid medium of IPTG and X-gal, wherein solid culture
The base benzyl mycin of ammonia containing 50mg/L；
E. coated plate is just being put in 5min in 37 DEG C of insulating boxs, then is inverted culture 12h and grows monoclonal；
(4) identification of recombinant clone
The multiple monoclonals of picking are in 4mL LB liquid medium, wherein fluid nutrient medium ammonia containing 50mg/L benzyl mycin, 37 DEG C of vibrations
Culture 12h is swung to bacterium solution muddiness, is extracted plasmid and is sequenced.
3. the gene with multiple DNA fragments high efficiency assemble method of zero background according to claim 1, which is characterized in that step 2 tool
Body is divided into following steps:
(1) CATP plasmid is the reading frame sequence of chloramphenicol resistance gene, does not include the starting of initiation codon ATG and upstream
Subsequence, gene both ends are two fermentoid restriction enzyme sites, and the segment cohesive end formed after two fermentoid BsaI digestions is respectively GCTT
And ACGG, cohesive end ACGG are connected with the carrier puc57-CATP after BsaI digestion；
(2) using pACYC-Duet1 as template, with primer CATP-PF/CATP-PR amplified fragments, obtained PCR fragment glue recycling
The pUC57 carrier of flush end connection EcoRV linearisation afterwards；
(3) connection product converts Escherichia coli TOP10 competent cell；
(4) identification of recombinant clone.
4. the gene with multiple DNA fragments high efficiency assemble method of zero background according to claim 1, which is characterized in that step 3 tool
Body is divided into following steps:
(1) target fragment is divided into small fragment, and adds two fermentoid sites
Target gene is divided into small fragment according to length without two fermentoid site BsaI, target gene, designs head and the tail primer amplification small pieces
Section, or by the small fragment of gene chemical synthesis synthesis target gene, two sites fermentoid BsaI and cohesive end are added in gene both ends, more
The cohesive end design principle of a genetic fragment are as follows: first 5 ' cohesive end of segment is consistent with linearized vector head, is
GAAT；3 ' cohesive ends of the last one segment are consistent with linearized vector tail portion, are GCTT；The 4bp's of different genes segment
Cohesive end is different, mutually not reverse complemental, avoids the occurrence of in 4 bases there are continuous 3 bases are identical or reverse complemental,
(2) small fragment is connected to cloning vector
After the PCR product glue recycling of several small fragments, it is connected respectively to the pUC57 carrier of EcoRV linearisation；
(3) connection product converts Escherichia coli TOP10 competent cell
(4) identification of recombinant clone.
5. the gene with multiple DNA fragments high efficiency assemble method of zero background according to claim 1, which is characterized in that step 4 tool
Body is divided into following steps:
(1) interior configuration group reaction cartridge is managed in 0.2mLPCR according to following system,
(2) after the completion of reaction system configuration, 0.2mL PCR pipe is placed in PCR instrument and covers rubber pad, according to following PCR
Response procedures are arranged in reaction condition.
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| CN114250241A (en) * | 2021-12-29 | 2022-03-29 | 上海英基生物科技有限公司 | One-step BsaI enzyme digestion connecting fragment assembling method, assembling kit and application |
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