KR101768770B1 - Positive screening method in yeast for the synthetic genes without frameshift and nonsense mutations - Google Patents
Positive screening method in yeast for the synthetic genes without frameshift and nonsense mutations Download PDFInfo
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Abstract
The present invention is intended to selectively select clones that do not contain an unintended transcriptional modification base error, that is, base deletion and insertion and termination codon mutation, in gene synthesis for protein expression.
To this end, a synthetic gene was inserted between the DNA binding site (LexA DBD, LexA DNA Binding Domain) of E. coli and the VP16, a transcription activation protein, in the E. coli-Yeast shuttle vector prepared in the present invention, VP16 is produced only in the absence of a restriction enzyme and ligase and in vivo cloning of the yeast and when there is a gene that does not have a transcriptional and termination mutation. Thus, an operator that binds to LexA DBD in the host yeast HIS3 and ADE2 reporter genes, including histidine and adenine auxotrophic host yeasts, can be grown in minimal medium to selectively clone synthetic genes and select genes with no transcriptional and termination codon mutations Method.
Description
The present invention relates to a method for selectively synthesizing a synthetic gene having a nucleotide sequence that is free of unintended base deletion, insertion and termination codon error during artificial gene synthesis for specific protein expression using yeast, ). ≪ / RTI >
Genetic information of living organisms can be extracted by using genetic engineering techniques as a common property of human beings. Gene synthesis technology is a technique for chemically synthesizing DNA of a gene or a dielectric length. It is distinguished from the synthesis of a common oligonucleotide in that it synthesizes DNA at a gene level of 1 kb or more at a high speed, high accuracy and low cost. Synthetic gene technology is difficult to obtain genes, inducing gene mutation at specific sites, producing chimeric genes, introducing and eliminating specific restriction enzyme sites, optimizing promoter sequences, converting proteins with low expression levels into appropriate codons in the expression system It is useful when the amount of expression is to be increased.
At present, a large number of oligonucleotides are required for artificially synthesizing genes of 1 kb or more in vitro . Oligonucleotides generally have about 40 to 50 bases per molecule based on the nucleotide sequence of the gene to be synthesized. Also, the oligonucleotides containing the adjacent nucleotide sequences have about 20 mutually complementary nucleotide sequences, and in particular, the cooling restoration temperatures should be the same. Oligonucleotides used in gene synthesis are designed to meet the above conditions and made by pure chemical methods.
An artificial synthetic gene of 1 kb or more using oligonucleotides can be produced by various methods, but it can be produced by assembly PCR, fusion PCR, and DNA ligase chain reaction Reaction method is used.
In many cases, the synthesis of an artificial gene is based on the error of the nucleotide sequence (especially the base deletion) occurring in the oligonucleotide synthesis process and the nucleotide sequence generated during the PCR process performed using the oligonucleotide, Finally, it should be identified and selected through sequencing. Synthetic genes can not be used even in the presence of a single nucleotide error, especially in the case of gene synthesis aimed at expression of proteins, a lack of nucleotides that modify the open reading frame or a termination codon within the insertion and decoding frame In the case of errors, the synthesized gene can not produce the correct protein.
To confirm the accuracy of current synthetic genes, the final DNA product is cloned and then relies entirely on sequencing analysis. The synthetic genes having different base sequences are discarded, and repeated synthesis is required until the correct nucleotide sequence is obtained. Therefore, much time, cost and effort are required.
Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and an object of the present invention is to provide a method for selectively synthesizing a synthetic gene having no deletion mutation (deletion and insertion of a base) It is intended to reduce the time, cost and effort used in the synthesis of the synthetic gene, differentiating it from the conventional method of sorting out the correct synthetic gene by relying entirely on a large number of sequencing analyzes.
In order to accomplish the object of the present invention as described above, the present invention provides a method for selectively selecting a synthetic gene free from a frame mutation and a non-sense mutation using a yeast and a recombinant vector.
In addition, the invention of the yeast genotype is MATa, trp1 -901, leu2 -3, 112, ura3 -52, his3Δ200, ade2, LYS2: :( lexAop) 4- HIS3, URA3: :( lexAop) 8- LacZ, And Leu2:: (lexAop) 8-ADE2 GAL4 .
In addition, the present invention provides a method for selectively screening synthetic genes, which is characterized by being the vector pGS-TRP of FIG.
The present invention is characterized in that said vector contains a TRP1, AMP R, ADH1 promoter, LexA DBD (DNA binding domain), VP16, the located between LexA and VP16 restriction enzyme Eco R1 recognition site, and a CYC1 terminator , And a method for selectively screening synthetic genes.
The present invention also relates to a method for producing a vector comprising the steps of: digesting said vector with a restriction enzyme Eco R1; Transforming the truncated vector and a linear synthetic gene into the yeast; Culturing the yeast in a minimal medium containing no nutrient tryptophan, histidine, or adenine (WHA) to select growing colonies; And examining the expression of LacZ having an operator capable of binding to the LexA DBD region of the protein expressed in the recombinant vector of said transformant; And a method for selectively screening a synthetic gene.
In this invention, it is possible to selectively screen only synthesized genes that have been completely eliminated when a deletion frame has been generated by base deletion and insertion in a synthetic gene for protein expression, or when a termination codon has been generated .
FIG. 1 shows electrophoresis results obtained by confirming the final product of gene A prepared by ligating 4-fragment DNA prepared by assembly PCR in the synthesis of gene A by Fusion PCR. The assembly PCR performed using three different polymerase enzymes, Dp (Doctor protein Pfu DNA polymerase), En (Enzynomic, npfu-Forte) and Ta (TaKaRa's PrimeSTAR HS Premix) and subsequent products of Fusion PCR (1,851 bp) and M is a size marker.
Fig. 2 shows the synthesis scheme of the gene A. Fig.
Figure 3 shows the results of gene cloning and sequencing of gene A using three different DNA polymerase A) Doctor protein Pfu DNA polymerase, B) Enzynomics npfu-Forte, C) TaKaRa PrimeSTAR HS Premix Lt; / RTI >
Figure 4 shows the pGS-TRP vector.
FIG. 5 shows the results of the simultaneous transformation of a synthetic gene having a nucleotide sequence complementary to the pGS-TRP vector at both ends and a pGS-TRP vector linearized with Eco RI into yeast, and two straight DNAs were recombined into a welcome DNA by Gap repair The transformant can be grown on a minimal medium in which tryptophan is omitted.
Figure 6 shows that histidine and adenine using the expression of the reporter gene with the LexA operator present in the host yeast were omitted and for the purposes of Figure 5 whether the growth in the minimal medium with the tryptophan omitted together and the expression of the LacZ reporter with the LexA operator Blue / white screening.
A) Transcriptional activity pattern of reporter gene in host by expression of fusion protein of LexA DBD-synthetic gene-VP16
B) Transformants selected from the minimal medium containing no WHA nutrient and X-gal
C) Blue colonies of transformants according to expression of LacZ in minimal medium supplemented with X-gal and no tryptophan and no transfectant growth on minimal media with no tryptophan, adenine and histidine and 1 mM 3-AT added
FIG. 7 shows the nucleotide sequence of the synthesized gene using the developed yeast system to determine whether the deletion or insertion of the synthetic gene resulted in deletion or insertion of the synthetic gene or that the termination codon was completely removed from the synthetic gene. ≪ / RTI >
Hereinafter, the present invention will be described in more detail with reference to Examples. However,
The embodiments of the present invention are provided only for the purpose of easier understanding of the present invention,
The present invention is not limited to the examples.
An embodiment of the present invention was carried out by the following conditions and methods.
Production of primers for artificial gene synthesis
Oligonucleotides were designed using Primer-BLAST ( http://www.ncbi.nlm.nih.gov/tools/ primer-blast) or Gene2oligo (http://berry.engin.umich.edu/gene2oligo/).
PCR and Assembly PCR
The PCR instrument used in this study was a Veriti thermal cycler from Applied Biosystems. PrimeSTARⓡ HS Premix (cat # R040A) from TaKaRa was used for DNA polymerase. Assembly PCR was performed using Veriti thermal cycler from Applied Biosystems. DNA polymerase was purchased from PrimeStar® HS Premix (cat # R040A) from TakaRatk, nPfu-Forte DNA polymerase (cat # P410) from Enzynomics and Pfu DNA polymerase from Doctor protein # DR00402) were used.
Molecular Cloning and Sequencing
The enzyme used in this study was Enzynomics' restriction enzyme and DNA ligase (Cat # DP004S). Restriction enzymes were used to cleave the PCR product and the specific recognition site of the MCS of the vector. PCR products and vectors in linear form were ligated in the form of a loop using DNA ligase, transformed into E. coli (XL1-Blue) by electrophoresis and screened on an antibiotic (ampicillin 0.1 mg / ml) medium. The transformants were additionally confirmed to be of the size predicted using restriction enzymes.
Gel extraction was performed using the LaboPass ™ Gel and PCR kit (cat # CMA0112) from Kosmosingtech Co., Ltd. for the recovery of the PCR products confirmed by agarose gel. For the extraction of plasmid DNA from E. coli transformants, the LaboPass ( TM) Plasmid Mini kit (cat # CMP0112) from Cosmos Tec Co., Ltd. was used. DNA sequence sequencing was commissioned by Cosmosin Tech. The sequencer was Applied Biosystems 'Automatic Sequencer ABI 3730xl and the sequencing reaction was Applied Biosystems' BioDye TM Terminator version 3.1.
Yeast transformation
The yeast used in this study was inoculated into 10 ml of YPD liquid medium, shake cultured (30 ° C., 200 rpm, 12 hr), transferred to a new YPD liquid medium and cultured to an
LYS2: :( lexAop) 4 - HIS3 , URA3: :( lexAop) 8 - LacZ, ADE2: :( lexAop) 8 - ADE2 GAL4
X-gal medium to confirm LacZ expression
To 800 ml of distilled water, add 1.7 g of YNB, 5 g of ammonium sulfate and 20 g of agar. Add 50 ml of 20X dropout solution and 100 X of stock solution as necessary. After sterilization, 50 ml of 40% glucose and 4 ml of X-gal solution were added to the minimal medium.
X-gal stock solution: After dissolving 5-bromo-4-chloro-3-indolyl-β-galactopyranoside (X-gal, USB) in N, N-dimethylformamide (DMF, Sigma) at a concentration of 20 mg / , Wrapped in foil and stored at -20 ° C.
10X BU salt solution (pH 7.0): 30 g of Na 2 HPO 4 and 70 g of NaH 2 PO 4 were sterilized by dissolving in 1 L of distilled water and stored at room temperature.
Example 1. Synthesis of 1,851 bp long gene A
As an example, to synthesize the DNA of gene A composed of nucleotides of about 1.8 kb, 1.8 Kb target gene was divided into 4 parts composed of about 500 bp and first synthesis of 4 pieces was performed using assembly PCR.
For the gene synthesis of each fragment, 30-50 bp oligonucleotides prepared for each fragment were added and PCR was performed (pre-denaturation at 95 ° C for 3 minutes, denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30
The four products of each fragment synthesized by Assembly PCR have the same nucleotide sequence in the adjacent fragment and terminal region. To synthesize the gene A of 1.8 Kb by connecting these four fragments, the 5 'end of gene A and 3 'Fusion PCR was performed using complementary oligonucleotide primer set. For fusion PCR, 25 cycles of pre-denaturation at 95 ° C for 3 minutes, denaturation at 95 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes, and final extension at 72 ° C for 5 minutes. The final product of Fusion PCR was confirmed on 1% agarose gel and is shown in FIG. A schematic diagram for the synthesis of 1.8 kb gene A is shown in Fig.
Example 2. Routine cloning and sequencing of the synthesized gene A
The gene A product synthesized as a result of Example 1 is linearized with pBluescript II-SK (pBS) using a restriction enzyme ( Xba I / Bam HI) with bases complementary to each other. Then, DNA ligase reaction was performed, and E. coli was transformed by electric shock. Transformants were selected on the medium containing Ampicillin antibiotics using Amp r , the antibiotic resistance gene of pBS, and the transformants were screened with 1% agarose gel after restriction enzyme Eco RI treatment.
Sequencing primers T3 and T7 were used to identify the nucleotide sequences. Analysis of the nucleotide sequence results was analyzed using the online tool ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Table 2 shows the types of base errors and the error rates in the synthesis of gene A using DNA polymerase from three companies, Doctor protein Pfu DNA polymerase, Enzynomics npfu-Fort and TaKaRa PrimeSTAR HS Premix. The error rate is calculated as error base / total base number.
Example 3: Using yeast in vivo cloning Decipherment frame Mutation and termination codon mutation Selective cloning and sequencing of missing clones
In this example, in vivo cloning of a synthetic gene using yeast was performed, and selection of a synthetic gene that did not include a deletion conformational error and a termination codon error was performed simultaneously.
Was it produced the pGS-TRP vector in order, pGS-TRP has an Amp r of antibiotic resistance markers in the yeast auxotrophic marker TRP1 as E. coli, including 2u the yeast replication origin is self allow replication in yeast cells . LexA DBD :: VP16 was inserted so that the DNA binding site (LexA DBD) of E. coli LexA and the VP16 protein capable of inducing transcriptional activation could be fused and expressed, which was regulated by the ADH1 promoter and the CYC1 terminator. For straightening of the pGS-TRP also it was inserted into the Eco RI restriction enzyme recognition site in order to insert the synthetic gene between the LexA DBD and VP16. The pGS-TRP vector is shown in Fig.
When the fusion PCR was carried out in the production of the synthetic gene in Example 1, about 25 base sequences complementary to each other based on the EcoRI of the pGS-TRP vector were added to the primers at both ends, Was prepared so as to have both ends of the gene A synthesized with pGS-TRP and a complementary base sequence.
The pGS-TRP digested with restriction enzyme Eco RI and the artificial synthetic gene of a straight line are simultaneously transformed into yeast, and gap repair is induced by using homologous base sequence of two products. The recombination is stabilized in the yeast by ligating two linear DNAs in a loop, and grows in the minimal medium due to the expression of the TRP1 gene present in the pGS-TRP vector. A schematic diagram thereof is shown in Fig.
The pGS-TRP vector, which was linearized with the synthetic gene DNA product and Eco RI, was transformed with the yeast host by the LiAc method at the same time and cultured in a minimal medium containing no tryptophan (W), adenine (A) and histidine Were selected.
In order for the transformants to grow on the minimal medium, i) two DNAs that are linearized in transformation are converted into circular recombinant DNA by homologous recombination and stabilized in the yeast to express the TRP1 gene, which is a selectable marker of pGS-TRP And ii) the recombinant synthetic gene does not contain a translation frame mutation, so that the fusion protein of LexA DBD-synthetic gene-VP16 should be made normally.
The fusion protein of LexA DBD-synthetic gene-VP16 is able to induce the expression of the reporter genes ADE2 and HIS3, which have an operator capable of binding to LexA inserted in the yeast, and grow on the minimal medium with nutrient WHA excluded. In particular, a minimal medium supplemented with 1 mM 3-AT (3-Amino-1,2,4-triazole) was used to supplement the leaky traits of host histidine auxotrophic mutations and to prevent false positive selection . The transfectants expressed the fusion protein of LexA DBD-synthetic gene-VP16 and eliminated false positives by investigating the expression of LacZ (β-galactosidase), another reporter to which this protein could bind. Expression of the LacZ reporter gene can be screened in blue / white using X-gal added to the medium, and degrades X-gal to blue color. The above procedure is shown in Fig.
The nucleotide sequences of the synthetic genes contained in the selected yeast transformants were determined using the primers SeqF and SeqR, and a total of 50 gene A nucleotide sequences were analyzed by Cosmogin Tech. The analysis was performed using the online tool ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and is shown in FIG.
The error rate is calculated as the error base / total base number. Synthetic gene identification by routine cloning as shown in Example 2 showed an error rate of 8.6 × 10 -4 with 40 errors out of 46, 575, and the error rate of artificial synthetic gene error developed in this study using yeast in Example 3 The elimination system confirmed the error rate of 1.7 × 10 -4 with 16 errors out of 92,550.
The base errors included in the clones synthesized by routine cloning in Example 2 include many defects and insertion errors that are the most problematic in oligosynthesis, while the synthetic synthesis using the developed yeast system No genetic clones were identified for deletion, insertion errors and nonsense mutations.
Sequence determination The success rate of the synthetic gene was calculated based on the number of clones (Colony). The success rate was calculated as a success / confirm clone. Five out of 25 synthetic clones selected by routine cloning showed a 20% success rate with error-free clones, and 38 of the 50 synthetic clones synthesized using the developed yeast were clones with no errors %, And analysis and comparison of synthetic gene errors using the developed yeast system are shown in Table 3.
Claims (5)
The genotype of the yeast is MATa, trp1-901, leu2-3, 112, ura3-52, his3Δ200, ade2, LYS2 :( lexAop) 4-HIS3, URA3: lexAop) 8-ADE2 GAL4, and
Wherein said recombinant vector comprises a TRP1, AMP r , ADH1 promoter, LexA, VP16, a restriction enzyme EcoR1 recognition site located between LexA and VP16, and a CYC1 terminator.
Wherein said vector is pGS-TRP of FIG. 4.
The selective selection of the synthetic gene
Digesting the recombinant vector with a restriction enzyme Eco R1;
Transforming the truncated vector and a linear synthetic gene into the yeast;
Culturing the yeast in a minimal medium containing no nutrients of tryptophan (W), adenine (A) and histidine (H) to grow colonies; And
Examining the expression of LacZ capable of binding to the protein expressed in said transformant; ≪ / RTI >
Selective selection of synthetic genes.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6682885B1 (en) | 1996-10-09 | 2004-01-27 | Srl, Inc. | Method for detecting mutations using an URA3 reporter gene |
| US20100297642A1 (en) | 2007-11-02 | 2010-11-25 | Geneart Ag | Method for determining frameshift mutations in coding nucleic acids |
| WO2010143871A2 (en) | 2009-06-08 | 2010-12-16 | 한국생명공학연구원 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6682885B1 (en) | 1996-10-09 | 2004-01-27 | Srl, Inc. | Method for detecting mutations using an URA3 reporter gene |
| US20100297642A1 (en) | 2007-11-02 | 2010-11-25 | Geneart Ag | Method for determining frameshift mutations in coding nucleic acids |
| WO2010143871A2 (en) | 2009-06-08 | 2010-12-16 | 한국생명공학연구원 | Method for screening and quantifying various enzyme activities using a genetic enzyme screening system |
Non-Patent Citations (2)
| Title |
|---|
| Am. J. Pathol., Vol. 159, No. 4, pp. 1239-1245 (2001.10.)* |
| Protein Expr Purif. 2013 Apr;88(2):235-42 |
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