KR101920036B1 - The screening method for gene without frameshift mutation and nonsense mutation using E.coli and ampicillin resistance gene - Google Patents

Abstract

The present invention relates to a plasmid vector for screening a gene without a frameshift mutation and a nonsense mutation using E.coli and an ampicillin resistance gene, and a method for screening a gene without a frameshift mutation and a nonsense mutation using the same. Because the plasmid vector has an antibiotic resistance gene marker which can be continuously expressed in addition to the ampicillin resistance gene, antibiotics can be firstly screened for transformants, and secondarily, an artificial gene synthesized not to have the frameshift mutation and the ampicillin resistance gene can be screened through the expression of the ampicillin resistance gene. Wherein, the ampicillin resistance gene can be expressed in association with the synthesized artificial gene by removing initiation codon.

Description

[0001] The present invention relates to a method for screening for a gene having no deletion, mobility, and terminal mutation using E. coli and an ampicillin resistance gene,

The present invention relates to a plasmid vector for selecting a gene having no transcriptional and translocation mutations using E. coli and an ampicillin resistance gene, and a method for selecting a gene having no transcriptional and translocation mutations using the plasmid vector.

Artificial DNA or gene synthesis technology refers to the technique of synthesizing short-length oligonucleotide fragments into genes of more than 1 kb using chemical and biochemical synthesis methods. Artificial gene synthesis is useful for synthesizing a specific protein but difficult to obtain template DNA, or as a means for improving a known protein to a new form or function. For example, when it is desired to induce mutation of a specific gene region and chimeric gene production, introduction and elimination of specific restriction enzyme sites, optimization of promoter arrangement, and expression of proteins with low production yields, Synthesis of genes is necessary.

In order to synthesize a target artificial gene in vitro, a single strand oligonucleotide having a length of 20 to 50 bp is required, and the step of combining these sequences is repeated to synthesize the entire sequence of the designed artificial gene as intended. Each oligonucleotide fragment produced by the pure chemical method is designed so that about 20 bases and a neighboring fragment can bind as a complementary sequence. Designed fragments should be connected to each other through a complementary sequence to form a single chain. For this purpose, the annealing temperature of all the oligonucleotide fragments set in the binding reaction (PCR: polymerase chain reaction) is the same . The method of synthesizing an artificial gene using PCR can be classified into an assembly PCR (assembly PCR) step, a fusion PCR step, a linking reaction by a DNA ligase, and a cloning step have.

One of the most important things in the synthesis of artificial genes is that a gene with a complete sequence without a mutation among synthetic genes of 1 kb or more should be produced as a final product.

In the synthesis of artificial genes, various sequence errors may occur. Errors in the sequence of nucleotides are inevitably involved in the synthesis of single-stranded oligonucleotides, assembly PCR and fusion PCR. Therefore, in order to determine the final synthetic synthetic gene after the cloning step, a process of identifying and selecting a synthetic gene having an accurate base sequence is essentially performed. When a protein is synthesized from an artificial gene containing a base error, the substitution of the base as a result of the base error may cause a change in the amino acid or a stop codon in the codon, and the deletion or insertion of the base may result in a DNA codon decoding reading frame, ORF), resulting in an amino acid change or a stop codon, which makes it impossible to produce an accurate protein. Therefore, an artificial synthetic gene should be exactly 100% identical to the designed sequence.

Currently, the confirmation of the accuracy of general synthetic genes depends entirely on the cloning of the final DNA product and the random base sequence analysis, and the artificial gene synthesis process and sequencing are repeatedly performed until a synthetic gene of the correct base sequence is obtained. to be. For this purpose, studies were carried out to identify transcriptional mobility and termination mutations in the synthesis of gene sequences and PCR. Korean Patent Laid-open Publication No. 10-2017-0015607 prepared a plasmid vector capable of inserting a synthetic gene between the DNA binding site of E. coli Lexa protein and the transcription activation protein VP16 present in E. coli-yeast shuttle vector, A technique has been known in which VP16 is produced only in the presence of a gene and histidine and an adenine auxotrophic host yeast are grown in a minimal medium so that mutation of the synthetic gene can be selected at the yeast stage. In addition, a method of confirming whether the expression level of a protein is changed by mutation using an expression vector prepared to include GFP as a reporter gene at an E. coli level (Non-patent Document 002) is known (see Non-Patent Document 002) Has been known as a plasmid vector capable of regulating the expression of a reporter gene depending on the mutation (Non-Patent Document 003). However, the above-described method for confirming whether a translation frame mutation or a termination mutation at an E. coli level is required requires a step of confirming the expression level or activity level of a reporter gene after separating one clone, and a randomly selected clone May be advantageous in that it can perform sequence analysis after confirming the expression level or activity level of a reporter gene, but it may still have time and cost-consuming limitations.

Accordingly, the present inventors have made efforts to develop a method for efficiently selecting only synthetic genes that do not have a framehift mutation and a nonsense mutation, which may occur due to nucleotide sequence variation in the process of synthesizing an artificial gene As a result, a plasmid vector capable of expressing the protein was prepared by linking the ampicillin resistance gene with the initiation codon removed to the terminal sequence of the synthetic gene. The plasmid vector contains a kanamycin antibiotic resistance gene and confirms the resistance of ampicillin to the first screened transformant to identify the artificial synthetic gene that is synthesized without the deletion frame movement mutation and termination mutation. The present inventors have completed the present invention.

KR 10-2017-0015607E (2017.02.09) US 6,284,496 B1 (November 4, 2001) US 9,155,390 B2 (Aug. 25, 2015)

 Akihiko Kataoka, et. al., American Journal of Pathology, Vol. 159, No. 4, October 2001.

As described above, it is required to synthesize an artificial synthetic gene so as not to have a translation frame mutation and a termination mutation which may occur due to addition, substitution or deletion of a base sequence in the step of synthesizing an artificial synthetic gene. It can be time consuming and expensive to screen.

Accordingly, it is an object of the present invention to provide a plasmid vector for gene selection which is free from a frameshift mutation and a nonsense mutation.

It is another object of the present invention to provide a gene selection method that does not have a translation frame movement mutation and a termination mutation.

In order to achieve the above object, the present invention provides a plasmid vector for screening a gene without a frameshift mutation and a nonsense mutation, which comprises the following (a) and (b):

(a) a gene cassette in which the Tac promoter and the ampicillin resistance gene (AmpR) from which the initiation codon has been removed are linked by a multiple cloning site (MCS) and a linker sequence; And

(b) Resistance genes for antibiotics except ampicillin.

In a preferred embodiment of the present invention, the restriction enzyme of (a) may be any one selected from the group consisting of BamHI, EcoRI, HindIII, SmaI and KpnI.

In a preferred embodiment of the present invention, said antibiotic (b) is selected from the group consisting of kanamycin, spectinomycin, streptomycin, bleomycin, erythromycin, polymyxin B, tetracycline, and chloramphenicol.

In a preferred embodiment of the present invention, the gene without the translation frame mutation and the termination mutation may be any one or both of an artificially synthesized gene and an organism-derived recombinant gene.

In a preferred embodiment of the present invention, the plasmid vector is lacI Gene. ≪ / RTI >

In a preferred embodiment of the present invention, the plasmid vector may be represented by the vector map shown in Fig.

In addition, the present invention provides a method for screening a gene free of a translation frame movement mutation and a termination mutation, which comprises the following steps i) to v):

i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of the present invention;

ii) transforming the host cell with the recombinant expression vector prepared in step i);

iii) culturing the transformant transformed in step ii) in an antibiotic marker-containing medium to induce colony formation;

iv) culturing the colonies formed in step iii) in a medium containing ampicillin and IPTG (Isopropyl? - D-1-thiogalactopyranoside) to induce colony formation; And

v) obtaining a transformant of the colony formed in step iv) above.

In addition, the present invention provides a method for screening a gene without transfection mobility and termination mutations, which comprises the following steps i) to iv):

i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of the present invention;

ii) transforming the host cell with the recombinant expression vector prepared in step i);

iii) culturing the transformant transformed in step ii) in a medium containing ampicillin and IPTG to induce colony formation; And

iv) obtaining a transformant of the colony formed in step iii) above.

In a preferred embodiment of the present invention, the antibiotic in step iii) may be kanamycin.

In a preferred embodiment of the present invention, the host cell of step ii) may be E. coli.

In a preferred embodiment of the present invention, the gene without the translation frame mutation and the termination mutation may be any one or both of an artificially synthesized gene and an organism-derived recombinant gene.

Accordingly, the present invention provides a plasmid vector comprising an ampicillin resistance gene with the start codon removed and a Tac promoter. Since the plasmid vector has an antibiotic resistance gene marker which can be continuously expressed in addition to the ampicillin resistance gene, the antibiotic can be firstly screened for the transformant, and secondarily, the expression of the ampicillin resistance gene And synthesized artificial genes so as not to have a termination mutation can be selected. Here, the ampicillin resistance gene can be expressed in association with a synthetic artificial gene by removing the initiation codon.

In addition, in the method of selecting a gene having no translation frame mutation and termination mutation through the plasmid vector of the present invention, it is expected that significant selection can be made even when only ampicillin is used as a selection marker. At this time, the synthetic gene is transformed with a recombinant plasmid vector so as to be linked with the ampicillin resistance gene from which the initiation codon has been removed, and the transformant can be directly cultured on a medium containing ampicillin and IPTG and cultured to select an antibiotic , It is possible to select only transformants having the synthetic gene so that they do not have a translation frame mutation and a termination mutation through the formation of colonies.

That is, when a synthetic artificial gene is inserted into a plasmid vector according to the present invention, a synthetic artificial gene having no translation frame mutation and terminal mutation can be selected through a simple step of culturing in an antibiotic-containing medium, The screening efficiency of the synthetic artificial gene is higher than that of the method, and the cost and time required for the research can be saved, which is effective.

1 is a schematic diagram showing a process of assembly PCR and fusion PCR for producing an artificial synthetic gene in the present invention.
Figure 2 shows the result of confirming synthetic gene A synthesized through assembly PCR and fusion PCR.
Figure 3 shows a vector map of plasmid vectors for gene selection without frameshift mutation and nonsense mutation according to the present invention.
FIG. 4 is a schematic diagram showing a process showing the resistance or sensitivity of amphicillin according to mutation when a synthetic gene is inserted into the plasmid vector of the present invention.
FIG. 5 shows the result of confirming the utility of the plasmid vector of the present invention using Aspergillus nidulans transcription factor A (about 1 kb).
FIG. 6 shows the process of confirming the utility of the plasmid vector of the present invention using an artificial synthetic gene A: FIG. 6A is a schematic diagram for inserting an artificial synthetic gene A into a vector, FIG. 6B shows a recombinant And E. coli transformed with the plasmid vector were cultured in a culture medium containing kanamycin.
FIG. 7 shows the results of confirming the utility of the plasmid vector of the present invention using an artificial synthetic gene A. FIG.
FIG. 8 shows a sequence analysis result of an artificial synthetic gene A in a total of 50 clones selected first in a kanamycin-containing medium using the plasmid vector of the present invention.
9 shows the gene A nucleotide sequence and mutation positions in clones (total 14) selected secondarily in the medium containing ampicillin and IPTG using the plasmid vector of the present invention.

Hereinafter, the present invention will be described in detail.

As described above, in the method for selecting a gene that does not have a translation motif and termination mutation that can occur in a synthetic gene or a recombination process, the gene selection method according to the prior art requires a research time There is a disadvantage in that there is a delay and a cost can be incurred.

Accordingly, the present invention provides a plasmid vector for gene selection free of frameshift mutation and nonsense mutation comprising (a) and (b):

(a) a gene cassette in which the Tac promoter and the ampicillin resistance gene (AmpR) from which the initiation codon has been removed are linked by a multiple cloning site (MCS) and a linker sequence; And

(b) Resistance genes for antibiotics except ampicillin.

In the " plasmid vector for gene selection " of the present invention, the gene may be any one or both of an artificial synthetic gene synthesized artificially and an organism-derived recombinant gene. More specifically, the artificial synthetic gene is synthesized by assembling and / or fusion of a fragment oligonucleotide for the purpose of obtaining a template DNA of a desired target or improving a novel form or function from a known protein Quot; means an artificial gene sequence. In addition, the recombinant gene refers to a gene sequence that is overexpressed in a plasmid vector for the purpose of overexpressing a known gene sequence or a gene sequence in which some sequences are substituted therefrom. When the recombinant gene is cloned to be overexpressed in the plasmid vector according to the present invention, there is a possibility that some gene base sequences may be mutated in the course of PCR amplification or the like in the cloning process. When the recombinant gene is cloned to overexpress in the plasmid vector according to the present invention, The gene product can be effectively screened.

In the "plasmid vector for screening a gene" of the present invention, the restriction enzyme of (a) may be any one or more selected from the group consisting of BamHI, EcoRI, HindIII, SmaI and KpnI, Any restriction enzyme that can be used in the cloning process can be selected and applied without limitation.

In the "plasmid vector for screening a gene" of the present invention, the antibiotic (b) may be kanamycin, spectinomycin, streptomycin, bleomycin, erythromycin, Polymyxin B, tetracycline and chloramphenicol. It is more preferable to use ganamycin, but it is not limited to this. Any antibiotic that can be used as a selectable marker in the course of research conducted using recombinant microorganisms can be selected and applied without limitation.

In the " plasmid vector for gene selection " of the present invention, the plasmid vector is lacI Gene, and more specifically, it is most preferably represented by the vector map shown in Fig. 1, but it is not limited thereto.

In a specific embodiment of the present invention, we first synthesized the artificial synthetic gene A through assembly PCR and fusion PCR (FIGS. 1 and 2). In addition, pET-TAmp having the sequence map of FIG. 3 was prepared to construct a plasmid vector for artificial synthetic gene selection that did not contain a framehift mutation and a nonsense mutation (FIG. 3 ). The pET-TAmp comprises a gene cassette in which the Tac promoter and the amphicillin resistance gene from which the start codon has been removed are linked to the linker sequence, and the kanamycin antibiotic resistance gene and the lactose operon inhibitory factor gene ( lacI ) Was prepared so that the inserted gene could be expressed in the medium.

In order to confirm whether or not the synthesized pET-TAmp can be usefully used, the present inventors firstly cloned and inserted the gene sequence derived from fungi in the pET-TAmp, and the colonies obtained in the kanamycin- And ITPG-containing medium, it was confirmed that colony growth was possible when the expression of protein was induced by lack of translation frame mutation and termination mutation (FIG. 4 and FIG. 5).

Further, the present inventors confirmed that pET-TAmp can also be used for an artificial synthetic gene (Fig. 6A). As a result, Escherichia coli transformed by inserting artificial synthetic gene A into pET-TAmp can form colonies in kanamycin medium (Fig. 6B). When cultured in the amphicillin + IPTG medium at random, it was confirmed that only clones having no mutation or terminal mutation could form colonies (Fig. 7).

In addition, the present inventors analyzed the sequence of artificial synthetic gene A in a clone forming colonies when cultured in an ampicillin + IPTG medium. As a result, a total of 14 clones did not contain translation frame mutation and termination mutation, It was confirmed that the sequence of artificial synthetic gene A was synthesized as a target sequence without any mutation (Fig. 8 and Fig. 9). On the other hand, it was confirmed that in all clones that do not form colonies in the ampicillin + IPTG medium, synthetic synthetic gene A having a translation frame mutation and / or a termination mutation was synthesized by substitution, deletion or insertion of a base sequence 8).

Therefore, when a synthetic artificial gene is inserted into the plasmid vector according to the present invention, a synthetic artificial gene having no translation frame mutation and terminal mutation can be selected through a simple step of culturing in an antibiotic-containing medium, The screening efficiency of the synthetic artificial gene is higher than that of the method, and the cost and time required for the research can be saved, which is effective.

In addition, the present invention provides a method for screening a gene without transfection mobility and termination mutations, which comprises the following steps i) to iv):

i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of the present invention;

ii) transforming the host cell with the recombinant expression vector prepared in step i);

iii) culturing the transformant transformed in step ii) in a medium containing ampicillin and IPTG to induce colony formation; And

iv) obtaining a transformant of the colony formed in step iii) above.

In addition, the present invention provides a method for screening a gene free of a translation frame movement mutation and a termination mutation, which comprises the following steps i) to v):

i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of the present invention;

ii) transforming the host cell with the recombinant expression vector prepared in step i);

iii) culturing the transformant transformed in step ii) in an antibiotic marker-containing medium to induce colony formation;

iv) culturing the colonies formed in step iii) in a medium containing ampicillin and IPTG (Isopropyl? - D-1-thiogalactopyranoside) to induce colony formation; And

v) obtaining a transformant of the colony formed in step iv) above.

In the " gene selection method " of the present invention, the gene may be any one or both of an artificial synthetic gene synthesized artificially and an organism-derived recombinant gene.

In the "gene selection method" of the present invention, the restriction enzyme of step ii) may be any one or more selected from the group consisting of BamHI, EcoRI, HindIII, SmaI and KpnI, And any restriction enzyme used or commercially available can be selected and applied without limitation.

In the " gene selection method " of the present invention, the host cell of step ii) is preferably Escherichia coli, but is not limited thereto and may be used as a host cell for gene recombination and over- Any cell can be used without limitation.

In the " gene selection method " of the present invention, the antibiotic of step iii) is selected from the group consisting of kanamycin, spectinomycin, streptomycin, bleomycin, erythromycin, It may be any one selected from the group consisting of polymyxin B, tetracycline, and chloramphenicol. More preferably, kanamycin is used, but it is not limited thereto, and recombinant Any antibiotic that can be used as a selectable marker in the course of research conducted using microorganisms can be selected without restriction.

In the method using the plasmid vector of the present invention, a synthetic artificial gene having no translation frame mutation and terminal mutation can be selected through a simple step of culturing in an antibiotic-containing medium. Therefore, the gene selection method of the present invention The screening efficiency of the synthetic artificial gene is higher than that of the method, and the cost and time required for the research can be saved, which is effective.

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

[Experimental Methods and Materials]

One. PCR  Way

In order to carry out the embodiment of the present invention, a veriti thermal cycler (Applied Biosystems) PCR instrument was used, and nPfu-Forte DNA polymerase (cat # P410, Enzynomics) having corrective activity was used as a DNA polymerase . The oligonucleotide used in the assembly PCR (PCR) experiment was a sequence designed using the online tool Gene2oligo (http://berry.engin.umich.edu/gene2oligo/). After PCR, the band of amplified PCR product was confirmed by electrophoresis, and the PCR product was recovered from the agarose gel using LaboPass ^™ Gel and PCR kit (cat # CMA0112, Kosomoguchi Inc.).

2. Cloning (cloning)

PBluescript II SK, pET-28a and pGEX-5T-1 plasmid vectors were used in the cloning process to produce the vector of the present invention. For cutting of the plasmid vector, Bam HI, Eco RI, Kpn I , and using the restriction enzymes Hin dIII (Enzynomics Co.) and, DNA T4 ligase (cat # DP004S, Enzynomics G) to connect the cut DNA or EZ -Fusion ™ Cloning Kit (cat # EZ015S, Enzynomics) was used.

The same restriction enzyme was used to cleave the PCR product and the specific recognition site of MCS in the vector (pBluescript II SK). The truncated linear forms of PCR products and vectors were ligated in circular form using DNA T4 ligase or cloning kit and transformed into E. coli XL1-Blue using electrophoresis. Transformed transformants were screened by inoculation on an agar medium containing antibiotics (ampicillin or kanamycin). EZ-Fusion ™ Cloning Kit (cat # EZ015S, Enzynomics) and Escherichia coli XL1-Blue strains were used for transformation using positive selection marker.

The selected transformants were inoculated on a liquid broth containing the same antibiotic and cultured, followed by centrifugation to obtain precipitated cells. Plasmid DNA was extracted from the obtained cells using LaboPass ^™ Plasmid Mini kit (cat # CMP0112, Kosomoguchi Inc.). The extracted plasmid DNA was digested with the same restriction enzyme as the restriction enzyme used in the above cleavage, and the size predicted by electrophoresis was confirmed.

3. Sequence analysis

Sequence analysis of the DNA product recovered after the PCR amplification or the plasmid DNA extracted from the transformant after cloning was carried out by Cosmosintech Co., At this time, an automatic sequencer ABI 3730xl (Applied Biosystems) was used as a sequencer, and BioDye ^TM Terminator version 3.1 (Applied Biosystems) was used as a sequencing program.

Synthesis of synthetic synthetic genes

First, as shown in the schematic diagram of FIG. 1, a gene A (gene A), which is an artificial synthetic gene sequence consisting of a nucleotide sequence of about 1.7 kb, was synthesized for use as a target sequence in the present invention.

In order to synthesize the gene A, first about 1.7 kb of the gene A sequence was divided into six parts so as to be a fragment of about 300 bp in length. The six regions were constructed so as to have the same base sequence so that each of the adjacent fragment regions and the end sequence could be connected to each other. To synthesize the respective fragments thus constructed, about 40 bp oligonucleotides were assembled by assemply PCR to synthesize a total of six product genes each having a length of about 300 bp (product A to product F ). Assembly PCR was performed in two stages of PCR. The PCR reaction conditions of the first step were pre-denaturation at 95 ° C for 3 minutes, denaturation at 95 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C The 30 sec procedure was repeated 5 times and the final-extension was performed at 72 ° C for 5 min to assemble the oligonucleotide of about 40 bp to a length of about 300 bp. A second step PCR was performed to amplify the assembled DNA. To this end, a primer complementary to the terminal region of each product gene was added to the PCR mixture and the PCR reaction was performed. The PCR reaction conditions were as follows: a pre-denaturation step of 95 ° C for 3 minutes, a denaturation step of 95 ° C for 30 seconds, a binding step of 58 ° C for 30 seconds, and an elongation step of 72 ° C for 30 seconds. For 5 minutes to finally obtain the assembled PCR product, which was then electrophoresed on 1% agarose gel to confirm the product (FIG. 2).

Fusion PCR was then performed to fuse each of the product genes synthesized by assembly PCR. A total of 6 gene products were mixed to prepare a PCR mixture. A forward primer complementary to the 5 'end and a complementary reverse primer to the 3' end were added thereto to amplify the gene A, Respectively. Fusion PCR was repeated 25 times for the pre-denaturation step of 95 ° C for 3 minutes, denaturation step of 95 ° C for 30 seconds, binding step of 58 ° C for 30 seconds, elongation step of 72 ° C for 30 seconds, and the final- Respectively. After the PCR reaction, the final product of the fusion PCR was confirmed by electrophoresis on 1% agarose gel (Fig. 2), and the identified gene A was extracted and obtained.

Decipherment frame  Migrating mutation frameshift  Plasmid vector construction for artificial synthetic gene selection without mutation and nonsense mutation

Using the transformation of microbial cells such as Escherichia coli, the plasmid vector of the present invention was produced to confirm whether or not the translation frame mutation and termination mutation occurred during the synthesis of an artificial synthetic gene. The plasmid vector was named pET-TAmp and was constructed to have a vector map of FIG.

First, the most basic pET-28a vector was prepared. The pET-28a vector contains the KanR gene, which is a gene resistant to kanamycin, which is an antibiotic against E. coli, and the lacI gene, which encodes an inhibitor of lactose operon. The T7 promoter, the multiple cloning site (MCS) and the T7 terminator portion were removed from the pET-28a plasmid.

The transgene was then obtained from the pGEX 5X-1 vector and pBluescript II SK for insertion at the protein expression partial site removed from the pET-28a plasmid. The Tac promoter gene in which the initiation of gene transcription was induced by IPTG from the pGEX 5X-1 vector was isolated, and the AmpR gene, which is an ampicillin resistance gene, was isolated from the pBluescript II SK vector and the AmpR (-ATG) Prepared. Then, a multiple cloning site (MCS) (SEQ ID NO: 1 '-GGATCCCCCGGGCTGGAATTC-3`) and a linker sequence (MCS) capable of introducing into the Tac promoter and the AmpR (-ATG) (SEQ ID NO: 2: 5 ' -GGAGCTGGTGCAGGCGCTGGAGCC-3 ') was inserted into the pET-28a plasmid in which the introduced gene was ligated and the protein expression site was removed.

Decipherment frame  Testing the utility of plasmid vectors for gene selection without migration and termination mutations

In order to test the utility of the plasmid vector prepared in Example 2, transcription factor A (about 1 kb) of Aspergillus nidulans , a fungal strain, was cloned into the pET-TAmp vector And then transformed into Escherichia coli XL1-Blue strain. Transformed transformants were first plated on LB agar medium (Kan broth) containing kanamycin and cultured. The transformant E. coli that produced the colonies in the blood culture medium was again cultured in LB agar medium (Amp medium) containing ampicillin; And LB agar medium (Amp + IPTG medium) containing ampicillin and IPTG, respectively, and then cultured, to confirm the formation of colonies.

In addition to the experimental group (pET-TAmp-gene A-cDNA) using the transcription factor A cDNA as an insert gene, a control (pET-TAmp) transformed with pET-TAmp without transcription factor inserted and a transcription factor A non- (PET-TAmp-gene A-gDNA) transformed with pET-TAmp into which genomic DNA containing the intron was inserted was used in combination with Kan, Amp, and Amp + IPTG To confirm the formation of colonies.

As a result, as shown in FIG. 5, the pET-TAmp control group exhibited resistance to antibiotics only in the kan culture medium to form colonies, and no resistance was observed in the Amp medium, so that no colonies were formed. In the case of the pET-TAmp-gene A-cDNA test group, it was confirmed that colonies were formed due to antibiotic resistance in Amp + IPTG medium containing IPTG that increases protein expression through the Tac promoter. In contrast, it was confirmed that no colonies were formed in the Amp medium without IPTG, and it was confirmed that resistance to ampicillin could not be shown due to suppression of protein expression. In addition, in the pET-TAmp-gene A-gDNA control group, the untranslated region was included in the inserted gene, and no Ampicillin resistance was observed in the Amp + IPTG medium as well as in the Amp medium. As a result, since the introns in the prokaryote, Escherichia coli, are induced to migrate, the ampicillin resistance protein can not be expressed from the AmpR gene having no initiation codon. As a result, the plasmid vector of the present invention has a gene The results of this study are summarized as follows.

Isolation and selective screening of mutant gene through plasmid vector with inserted synthetic gene sequence

When the pET-TAmp vector of the present invention was used, it was confirmed that transfectants that do not contain a translation-mutant and a termination mutation that can appear in gene synthesis and recombination can be selected, Sequence was inserted to reaffirm the screening efficiency.

First, the pET-TAmp vector prepared in Example 2 and the gene A prepared in Example 1 were digested with the same restriction enzymes, and then A was cloned into a pET-TAmp vector using an Ez-Cloning kit After insertion through homologous recombination (Fig. 6A), E. coli was transformed by electroporation. Transformed transformants were first plated on LB agar medium (Kan broth) containing kanamycin and cultured. Of the transformant E. coli (FIG. 6B) that produced colonies in the blood culture medium, 50 clones were randomly selected and isolated, and clones were inoculated again into Kan, Amp and Amp + IPTG media , And the formation of colonies was confirmed.

As a result, as shown in [Table 1] and Fig. 7, all 50 clones isolated from the Kan medium did not grow in Amp medium. It was also confirmed that 14 clones out of 50 clones grew on Amp + IPTG medium and 36 clones did not grow (Fig. 7).

Antibiotic resistance of transformants Base error Base sequence
Sum Number of successful clones synthesis
Success rate Antibiotic Resistance Number of clones defect insertion substitution Sum Blood
(Full clone) ○ 50 46 21 11 78 86,400 10 20% Amp
(+ IPTG) × 36 46 21 6 73 62,208 0 0% Amp
(+ IPTG) ○ 14 0 0 5 5 24,192 10 71.4%

Then, the nucleotide sequence of gene A was analyzed from 50 clones by DNA sequencing to confirm the mutation of the nucleotide sequence. For sequence analysis, a primer was prepared using the nucleotide sequence of the Tac promoter and the AmpR gene adjacent to the gene A.

As a result, as shown in Table 1, FIG. 8, and FIG. 9, it was confirmed that there were no base errors of gene A in 10 clones out of 14 clones growing in Amp + IPTG medium, Were found to have a missense mutation. On the other hand, all 36 clones that could not grow on Amp + IPTG medium showed mutation of transcriptional translation due to base insertion and / or deletion in gene A.

These mutations were analyzed to confirm the success rate of synthesis of the gene A synthesis step. In the present invention, the total number of bases of the gene A synthesized for use as the target nucleotide sequence is 86,400, 78 nucleotides are mutated, and 1 out of 1,108 nucleotides has a nucleotide sequence variation. When the frequency of base error was calculated through the variation of the nucleotide sequence among the 14 clones grown on the Amp + IPTG medium, 5 base mutations were found out of a total of 24,192 bases, and one of the 4,838 bases had a mutation of the base sequence As a result, the base error rate was effectively reduced.

That is, in the screening step of the transformant using only the kan medium, the synthesized clone was found to have a synthesis success rate of 20% in a total of 50 clones selected so as not to exhibit the translation frame mutation and the final mutation among the 50 clones randomly selected. And Amp + IPTG medium did not show translation frame mutation and termination mutation in 10 clones among 14 clones, indicating that the synthesis success rate of about 71.4% was obtained (Table 1).

<110> PAICHAI UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> The screening method for gene without frameshift mutation and          nonsense mutation using E. coli and ampicillin resistance gene <130> 1062521 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> multiple cloning site <400> 1 ggatcccccg ggctggaatt c 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> Linker sequence <400> 2 ggagctggtg caggcgctgg agcc 24

Claims (11)

(a) a Tac promoter and an amphicillin resistance gene (AmpR) from which the initiation codon has been removed are selected from a multiple cloning site (MCS) consisting of the nucleotide sequence of SEQ ID NO: 1 and a linker sequence consisting of the nucleotide sequence of SEQ ID NO: Linker sequence); And
(b) a pET-Tamp vector represented by the vector map shown in FIG. 3, which contains the resistance gene marker for kanamycin and the lacI gene;
Plasmid vectors for gene selection without frameshift mutation and nonsense mutation
[Figure 3]

.
The plasmid vector according to claim 1, wherein the restriction enzyme of (a) is any one selected from the group consisting of BamHI, EcoRI, HindIII, SmaI and KpnI.
delete 2. The plasmid vector according to claim 1, wherein the gene without the translation frame mutation and the termination mutation is either or both of an artificially synthesized gene and an organism-derived recombinant gene.
delete delete i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of claim 1;
ii) transforming E. coli with the recombinant expression vector prepared in step i);
iii) culturing the transformant transformed in step ii) in a culture medium containing kanamycin to induce colony formation;
iv) culturing the colonies formed in step iii) in a medium containing ampicillin and IPTG (Isopropyl? - D-1-thiogalactopyranoside) to induce colony formation; And
v) obtaining a transformant of the colony formed in the step iv); and selecting a gene without the deletion frame movement mutation and the termination mutation.
i) preparing a recombinant expression vector by inserting a target gene into the plasmid vector of claim 1;
ii) transforming E. coli with the recombinant expression vector prepared in step i);
iii) culturing the transformant transformed in step ii) in a medium containing ampicillin and IPTG to induce colony formation; And
iv) obtaining a transformant of the colony formed in the step iii); and selecting a gene without a translation frame mutation and a termination mutation.
delete delete 9. The method according to claim 7 or 8, wherein the gene without the translation frame mutation and the termination mutation is either one or both of an artificially synthesized gene and an organism-derived recombinant gene. Mutation-free gene selection method.

Images