CN117802133A - Method for improving synthesis efficiency of long genes - Google Patents
Method for improving synthesis efficiency of long genes Download PDFInfo
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- CN117802133A CN117802133A CN202311655840.7A CN202311655840A CN117802133A CN 117802133 A CN117802133 A CN 117802133A CN 202311655840 A CN202311655840 A CN 202311655840A CN 117802133 A CN117802133 A CN 117802133A
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 42
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 30
- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000012634 fragment Substances 0.000 claims abstract description 49
- 238000012216 screening Methods 0.000 claims abstract description 28
- 239000013598 vector Substances 0.000 claims abstract description 15
- 238000012163 sequencing technique Methods 0.000 claims abstract description 13
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000000746 purification Methods 0.000 claims abstract description 5
- 238000012795 verification Methods 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 22
- 230000003321 amplification Effects 0.000 claims description 17
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 17
- 238000012408 PCR amplification Methods 0.000 claims description 16
- 238000011901 isothermal amplification Methods 0.000 claims description 8
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- 235000016496 Panda oleosa Nutrition 0.000 claims description 6
- 240000000220 Panda oleosa Species 0.000 claims description 6
- 229960005091 chloramphenicol Drugs 0.000 claims description 6
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 6
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 229930027917 kanamycin Natural products 0.000 claims description 3
- 229960000318 kanamycin Drugs 0.000 claims description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 3
- 229930182823 kanamycin A Natural products 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
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- 230000011218 segmentation Effects 0.000 claims description 2
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- 101150113191 cmr gene Proteins 0.000 abstract description 12
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- 241000588724 Escherichia coli Species 0.000 abstract description 2
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for improving the synthesis efficiency of a long gene, which belongs to the technical field of gene synthesis and comprises the following steps: step one, segmenting genes; secondly, synthesizing 2-3kb intermediate fragment genes; thirdly, synthesizing the full length of the 10kb genes; realizes the rapid and efficient synthesis of 10k genes. And when the segments are recombined, a CmR gene segment is added, screening is carried out by using a double-antibody flat plate, the CI gene segment comprising the assembled segment cannot grow on the double-antibody flat plate, the vector which is not cut cleanly can not grow on the double-antibody flat plate, background pollution from the vector and the insert segment template is discharged, and the success rate of assembly screening is improved. In addition, the invention improves the flow of gene synthesis, utilizes phi29DNA polymerase to amplify and recombine single colony in the construction process of the intermediate fragment, and performs sequencing analysis after purification and is used as a template for next round of assembly, without culturing Escherichia coli, thereby saving time and cost.
Description
Technical Field
The invention belongs to the technical field of gene synthesis, and particularly relates to a method for improving synthesis efficiency of long genes.
Background
Modern molecular biology, genetic engineering and synthetic biology are increasingly demanding for long gene synthesis. In the field of molecular biology, researchers often need to redesign and construct plasmids larger than 10kb from scratch in order to express a protein or to explore the function of a certain pathway. Gene lines and metabolic pathways constructed by synthetic biological scientists often contain tens or even tens of origins, and the length of individual modules is often 10kb or even more than 20 kb. The Gibson recombination is a main means of gene synthesis gradually due to the simplicity and high efficiency, the recombination success rate of the existing recombinase on the market for 1-2 fragments can be more than 90%, and the recombination success rate is rapidly reduced along with the increase of the number of fragments and the increase of the fragment length.
Disclosure of Invention
The invention aims to provide a method for improving the synthesis efficiency of a long gene, which aims to solve the problem that the recombination success rate is rapidly reduced along with the increase of the number of fragments and the increase of the fragment length in the Gibson recombination process.
The invention provides a method for screening by adding more than one CmR gene segment and adopting a double-antibody flat plate, wherein the resistance of a target vector can be any resistance gene except the CmR resistance gene.
The aim of the invention can be achieved by the following technical scheme:
a method for improving the synthesis efficiency of a long gene, comprising the steps of:
first step, gene segmentation:
the 10kb gene fragment was designated as A, B, C, D for 4 2-3kb fragments, the A, B, C, D fragment was designated as Ab, ac, ad, ba, bb, bc, bd, ca, cb, cc, cd, da, db, dc, dd for 12 800-900bp fragments, and the CmR was amplified by PCR from pACYC184 vector.
And (3) synthesis of 800-900bp small segment genes:
12 800-900bp small segments are respectively designed into oligo with 15-20bp overlapped with each other through primer design software, and are recombined with a Puc57 vector (EcoRV) through PCA Polymerase Chain Assembly (PCA), introduced into DH5a competent cells, and subjected to colony PCR and amplification verification.
Second step, 2-3kb intermediate fragment gene synthesis:
fragment A synthesis: designing PCR amplification primers, carrying out PCR amplification by taking isothermal amplification products of Aa, ab and Ac with correct sequences as templates (20-50 bp recombination arms are introduced at two ends of Aa, ab and Ac), simultaneously adding a CmR fragment at the tail end of the A fragment, recombining with a Puc57-Kan vector (EcoRV), introducing DH5a competent cells, screening with a double antibody of kana and chloramphenicol, and carrying out colony PCR and amplification verification.
B. C, D fragment A was synthesized.
Third step, full-length synthesis of 10kb genes:
designing PCR amplification primers, carrying out PCR amplification by taking a A, B, C, D isothermal amplification product with correct sequencing as a template (20-50 bp recombination arms are introduced at two ends of A, B, C, D), simultaneously adding a CmR fragment (the head and the tail of the fragment are both introduced with a restriction enzyme site HindIII and a cloning site of pet-28a (+), recombining with a linearized pet-28a (+) vector, introducing DH5a competent cells, screening with a dual antibody of kana and chloramphenicol, culturing overnight at 37 ℃, carrying out colony screening PCR verification the next day, and picking and verifying correct clones for plasmid extraction, sequencing verification and enzyme digestion verification.
Further, in the third step, the correct clone is sequenced, the HindIII enzyme is used for cutting off the CmR fragment, T4DNA ligase is added to enable DNA to be self-connected, DH5a competent cell kanamycin flat plate is introduced for screening and culturing at 37 ℃ overnight, colony screening and PCR verification are carried out the next day, and the correct clone is selected for plasmid extraction, sequencing verification and enzyme digestion verification.
Further, colony PCR and amplification verification comprises the following steps: culturing overnight at 37 ℃, and carrying out colony screening PCR verification on the next day, wherein the corresponding single colony is amplified by phi29DNA polymerase for 6 hours; colony screening PCR verifies that positive colonies find isothermal amplification products, and sequencing and verifying after purification.
The invention has the beneficial effects that:
the invention provides a method for improving the synthesis efficiency of long genes, which realizes the rapid and efficient synthesis of 10k genes. During multistage recombination, a CmR gene fragment (not expressed) is added, only successfully assembled clones can express the second resistance (CmR), the single clones on the plate are all target clones in theory, the double-antibody plate is utilized for screening, the template from the assembled fragments cannot grow on the double-antibody plate, the vector which is not completely digested cannot grow on the double-antibody plate, the background pollution from the vector and the inserted fragment template is eliminated, and the success rate of assembly screening is improved.
The method improves the flow of gene synthesis, utilizes phi29DNA polymerase to amplify and recombine single colony in the construction process of the intermediate fragment, and performs sequencing analysis after purification and is used as a template for next round of assembly, thus the method does not need to culture escherichia coli and saves time and cost.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a PCA amplification assay of example 1 of the present invention via PCA polymerase chain assembly;
FIG. 2 is a chart showing the detection of PCA amplification gel electrophoresis in example 1 of the present invention;
FIG. 3 is a diagram showing colony screening PCR gel electrophoresis detection in example 1 of the present invention;
FIG. 4 is a diagram showing the electrophoresis detection of the amplification of a monoclonal colony by phi29DNA polymerase in example 1 of the present invention;
FIG. 5 is a diagram showing the detection of the intermediate fragment assembly PCR amplification gel electrophoresis in example 2 of the present invention;
FIG. 6 is a diagram showing the detection of intermediate fragment assembly colony screening PCR amplification gel electrophoresis in example 2 of the present invention;
FIG. 7 is a gene A-CmR full plasmid map of example 3 of the present invention;
FIG. 8 is a map of the whole plasmid of ene A in pET-28a (+) in example 3 of the present invention;
FIG. 9 is a PCR amplified gel electrophoresis detection chart of the assembled fragment of ABCD in example 3 of the present invention;
FIG. 10 is a diagram showing the electrophoresis of HindIII digested plasmid gene A-CmR in example 3 of the present invention;
FIG. 11 is a diagram showing the detection of the MluI and SmaI double digested plasmid gene A in pET-28a (+) in example 3 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The primer sequences used in the examples are shown in Table 1 below:
TABLE 1
The types and designations of reagents used in the examples are shown in Table 2 below:
TABLE 2
Example 1
800-900bp segment gene synthesis:
the 800-900bp segmented genes are respectively designed into oligo which are overlapped with each other by 15-20bp, the oligo is assembled by PCA polymerase chain, a PCA amplification detection diagram is shown in figure 1, the oligo is recombined with a Puc57 vector (EcoRV), TOP10F' competent cells are introduced, the cells are cultured for 14-18h at 37 ℃ overnight, colony PCR verification is carried out, phi29DNA polymerase amplification is carried out on corresponding single colonies at the same time, and amplified products are used as templates for sequencing verification and next round of assembly. Compared with the traditional method, the method does not need to carry out amplified culture and plasmid extraction on the monoclonal. PCA amplification experimental procedure:
1. preparing PCR reaction, preparing working solution, adding the working solution and the primer into a 96-hole PCR reaction plate (0.2 EP tube.0.2 eight-connecting tube), and detecting fragments after the reaction is finished; the PCR amplification system is shown in Table 3 below:
TABLE 3 Table 3
The PCR reaction procedure is shown in Table 4 below:
TABLE 4 Table 4
Monoclonal RCA Experimental procedure
1) Colonies were picked to 20. Mu. LddH 2 O was mixed and 1. Mu.l was used for the amplification reaction, the system is shown in the following table:
TABLE 5
| Reagent(s) | Volume of |
| Phi29MAXDNAPolymeraseReactionBuffer(10×) | 1μl |
| phi-p(100μM) | 2.5μl |
| Sample to be amplified | 1μl |
| ddH2O | 4μl |
| Total | 8.5μl |
2) Incubation was carried out at 95℃for 3min, immediately cooled on ice for 5min, the system being as shown in Table 6 below:
TABLE 6
3) Incubating at 30 ℃ for more than 6 hours;
4) Incubate at 65℃for 10min for inactivation. The PCA amplification gel electrophoresis detection diagram is shown in FIG. 2; colony screening PCR gel electrophoresis detection diagram detection 837bp is shown in figure 3; the map of the amplification of the monoclonal colony by phi29DNA polymerase is shown in FIG. 4; colony screening PCR verifies that positive colonies find isothermal amplification products, and sequencing and verifying after purification.
Ab, ac, ad, ba, bb, bc, bd, ca, cb, cc, cd, da, db, dc, dd was synthesized in the same manner.
Example 2
2-3kb intermediate fragment Gene Synthesis
Fragment A synthesis: designing PCR amplification primers, carrying out PCR amplification by taking Aa, ab and Ac isothermal amplification products with correct sequences as templates (20-50 bp recombination arms are introduced at two ends of Aa, ab, ac, ad), carrying out Gibson recombination with fragment 2 (CmR gene) and fragment 1 (linearization vector), introducing DH5a competent cells, screening with kana and chloramphenicol double antibodies, culturing overnight at 37 ℃, carrying out colony screening PCR verification the next day, carrying out phi29DNA polymerase amplification on corresponding single colonies at the same time, and taking the amplified products as templates for sequencing verification and next round of assembly.
Fragments B, C, D were synthesized in the same manner.
The PCR amplified fragments are shown in the following table 7, the intermediate fragment assembly PCR amplified gel electrophoresis detection is shown in the reference 5, and the intermediate fragment assembly colony screening PCR amplified gel electrophoresis detection 763bp is shown in the reference 6;
TABLE 7
Example 3
Complete gene assembly
PCR amplification primers were designed, PCR amplification was performed with the properly sequenced A, B, C, DRCA product as template (20-50 bp recombination arms were introduced at both ends of A, B, C, D), and simultaneously CmR fragments were added (HindIII unique recognition site was introduced at the head of the fragment in view of the presence of a HindIII unique recognition site at the end of the full plasmid sequence), gibson recombination was performed with NdeI and HindIII linearized pet-28a (+) vector, DH5a competent cells were introduced, kana and chloramphenicol double antibody selection, overnight incubation at 37℃and the next day colony screening PCR verification were performed, the correct clone was picked for plasmid extraction and restriction enzyme verification, and the full sequence map was shown in FIG. 7 (geneA-CmR full plasmid map), and the full sequence map was shown in FIG. 8 (geneAinpET-28 a (+) full plasmid map).
The correct clone was digested with HindIII to remove CmR fragment, and simultaneously T4DNA ligase was used to self-ligate linearized DNA, which was introduced into DH5a competent cell kanamycin plate for overnight incubation at 37℃and the next day of colony screening PCR validation, the correct clone was picked for plasmid extraction and sequencing analysis, and the full sequence map was shown in FIG. 8 (geneAinpET-28 a (+) full plasmid map). The detection of the ABCD assembled fragment by PCR amplification gel electrophoresis is shown in figure 9; the HindIII digested plasmid geneA-CmR electrophoresis detection diagram is shown in FIG. 10, the lane 1 in FIG. 10 is a plasmid, the lane 2 is HindIII digested plasmid geneA-CmR, and the theoretical occurrence band is 769bp and 13678bp; the MluI and SmaI double restriction plasmid geneAinpET-28a (+) electrophoresis detection diagram is shown in reference to 11, a lane 1 in the diagram is a plasmid, a lane 2 is the MluI and SmaI double restriction plasmid geneA-CmR, and the theoretical occurrence band is 3173bp and 11274bp;
PCR amplification is shown in Table 8 below:
TABLE 8
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for improving the synthesis efficiency of a long gene, comprising the steps of:
first step, gene segmentation:
the 10kb gene fragment was designated as A, B, C, D for 4 2-3kb fragments, and A, B, C, D as 12 800-900bp fragments, respectively, as Ab, ac, ad, ba, bb, bc, bd, ca, cb, cc, cd, da, db, dc, dd;
and (3) synthesis of 800-900bp small segment genes:
respectively designing 12 800-900bp small segments into oligo with 15-20bp overlapped with each other through primer design software, assembling with a Puc57 vector through PCA polymerase chain reaction, introducing DH5a competent cells, and performing colony PCR and amplification verification;
second step, 2-3kb intermediate fragment gene synthesis:
fragment A synthesis: designing PCR amplification primers, carrying out PCR amplification by taking isothermal amplification products of Aa, ab and Ac with correct sequences as templates, simultaneously adding CmR fragments at the tail ends of the A fragments, recombining with Puc57-Kan vectors, introducing DH5a competent cells, screening with double antibodies of kana and chloramphenicol, and carrying out colony PCR and amplification verification;
B. c, D fragment synthesis is the same as A;
third step, full-length synthesis of 10kb genes:
designing PCR amplification primers, carrying out PCR amplification by taking a A, B, C, D isothermal amplification product with correct sequence as a template, simultaneously adding a CmR fragment, recombining with a linearized pet-28a (+) vector, introducing DH5a competent cells, screening with a dual antibody of kana and chloramphenicol, culturing overnight at 37 ℃, carrying out colony screening PCR verification the next day, and picking and verifying correct clones for plasmid extraction, sequencing verification and enzyme digestion verification.
2. The method for improving the synthesis efficiency of long genes according to claim 1, wherein in the third step, the correct clone is sequenced, the HindIII enzyme is used for cutting off the CmR fragment, the T4DNA ligase is added to enable DNA to be self-linked, DH5a competent cell kanamycin flat plate screening is carried out at 37 ℃ for overnight culture, colony screening PCR verification is carried out the next day, and the correct clone is selected for plasmid extraction, sequencing verification and enzyme digestion verification.
3. The method for improving the synthesis efficiency of long genes according to claim 1, wherein colony PCR and amplification verification comprises the following steps: culturing overnight at 37 ℃, and carrying out colony screening PCR verification on the next day, wherein the corresponding single colony is amplified by phi29DNA polymerase for 6 hours; colony screening PCR verifies that positive colonies find isothermal amplification products, and sequencing and verifying after purification.
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