CN110607317A

CN110607317A - Method for regulating gene expression by using novel dCas9

Info

Publication number: CN110607317A
Application number: CN201910923497.7A
Authority: CN
Inventors: 霍毅欣; 郭丽微; 刘玉美
Original assignee: Suzhou Industrial Park Ucla Institute Of Advanced Technology; Beijing University of Technology
Current assignee: Suzhou Industrial Park Ucla Institute Of Advanced Technology; Beijing University of Technology; Beijing Institute of Technology BIT
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-24

Abstract

Since the CRISPRi system can block initiation or elongation of an RNA polymerase complex to inhibit gene transcription, the dCas9 gene is expressed after being linked to various inducible promoters in order to controllably regulate gene transcription. However, due to the loose regulation mechanism of some inducible promoters, dCas9 still has weak expression ability without adding an inducer, thereby partially inhibiting the target gene. In large-scale fermentation of industrial production, precise regulation and control of genes need to be realized, so the problem of leakage expression caused by an inducible promoter needs to be solved urgently. The invention aims to provide a method for regulating gene expression by using a novel dCas9 system, which utilizes the difference of codon preference and tRNA aminoacylation level to simply and effectively realize the expression regulation of dCas9 gene by adding amino acid at the translation level. The amino acid in the dCas9 gene is replaced by a rare codon form, and the corresponding amino acid is added by external sources, so that the protein with the expression function can be restored to play a role in inhibiting.

Description

Method for regulating gene expression by using novel dCas9

The technical field is as follows:

the invention relates to a method for regulating gene expression by using a novel dCas9 system, belonging to the technical field of biological engineering.

Background art:

the CRISPRi system can inhibit gene transcription by blocking initiation or elongation of an RNA polymerase complex, and thus is widely used for various microorganisms, cells, and the like. In general, the dCas9 gene is expressed in conjunction with various inducible promoters in order to controllably regulate the transcription of the gene. However, due to the loose regulation mechanism of some inducible promoters, the dCas9 still has weak expression ability without adding an inducer, thereby partially inhibiting the target gene. In large-scale fermentation of industrial production, precise regulation and control of genes need to be realized, so the problem of leakage expression caused by an inducible promoter needs to be solved urgently.

The amino acid in the dCas9 gene is replaced by a rare codon form, and the corresponding amino acid is added by external sources, so that the protein with the expression function can be restored to play a role in inhibiting. By this strategy, the expression of genes can be more precisely regulated at the translation level, reducing gene leakage expression; the inducer has no toxic or harmful effect on cells and is low in price; the rare codon only acts on the gene containing the codon to regulate the expression of dCas9 gene, and does not interfere with the expression of other genes and influence the normal growth of thallus.

The invention content is as follows:

the invention aims to provide a method for regulating gene expression by using a novel dCas9 system, which utilizes the difference of codon preference and tRNA aminoacylation level to simply and effectively realize the expression regulation of dCas9 gene by adding amino acid at the translation level.

According to the technical scheme provided by the invention, the method for regulating and controlling gene expression by using the novel dCas9 system comprises the following steps:

1. the strains used and their codon preference need to be determined.

2. According to the codon preference of amino acid and dCas9 gene sequence, the selected amino acid and its rare codon are determined, the codon of correspondent amino acid in the gene to be regulated and controlled is substituted by correspondent rare codon, and said sequence can be artificially synthesized again by using PCR method.

3. Connecting the gene sequence artificially synthesized in the step 2 with the plasmid vector 1, transferring the connecting product into a sensitive cell, selecting a correct single colony through PCR verification, storing a bacterial liquid, and extracting the plasmid 1.

4. Designing sgRNA targeting a gene required to be regulated, connecting a sgRNA expression frame to a vector 2 by utilizing PCR, transferring a connecting product into a sensitive cell, selecting a correct single colony through PCR verification, preserving bacterial liquid, and extracting a plasmid 2.

5. And (3) transforming the two plasmids extracted in the step (3) and the step (4) into the same competent cell, coating a flat plate, selecting a single colony, inoculating the single colony into a culture medium for culture, supplementing corresponding amino acid, and detecting the expression of the regulatory gene.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials, reagents, etc. used in the following examples are commercially available without specific reference.

The following examples are further illustrative of the present invention and are not to be construed as limiting the spirit of the present invention.

Example 1A method for regulating GFP expression Using the novel dCas9 System

For example, Escherichia coli DH5 α (available from Beijing Bomaide Gene technology, Ltd.), the leucine codons used for translation of Escherichia coli DH5 α have six types, UUG, UUA, CUC, CUG, CUU and CUA, in which the codon CUA is used with the lowest frequency (non-patent documents describing codon preference of Escherichia coli are Dong H, Nilsson L, Kurland C G.,1996, Co-variation of tRNA absorbance and code use in Escherichia coli at differential growth rates. journal of molecular biology,260(5): 649-. All codons corresponding to leucine in dCas9 are replaced by a rare codon CUA, and a method of gene total synthesis is adopted to obtain a mutant dCas9 protein gene.

The gene total synthesis system is as follows: 5 × TransStart O.RTFastPfFly Buffer 10. mu.L, dNTP (2.5mM) 4. mu.L, primer premix 2. mu.L, TransStart O.RTFastPffy DNA Polymerase 1. mu.L, distilled water 35. mu.L, total volume of 50. mu.L. The amplification conditions were denaturation at 95 ℃ for 20 seconds, annealing at 55 ℃ for 20 seconds, and extension at 72 ℃ for 20 seconds (30 cycles); extension at 72 ℃ for 5 min (1 cycle). The amplification product comprises the dCas9 protein gene and 515 bases upstream and 118 bases downstream of the dCas9 protein gene, and is connected to the carrier 1. A connection system: 1.5. mu.L of PCR amplification product, 1. mu.L of vector 1 fragment, 7.5. mu.L of GibsonMaster Mix (from NEW ENGLAND BioLabs), gently mixed, and reacted in a water bath at 50 ℃ for 60 minutes. Then 50. mu.L of DH 5. alpha. competent cells (purchased from Beijing Bomaide Gene technology Co., Ltd.) were added, and the mixture was immediately placed on ice for 2 minutes with ice bath for 30 minutes and heat shock at 42 ℃ for 60 seconds. Add 250. mu.L SOC medium and shake culture in a shaker at 200rpm and 37 ℃ for 1 hour. And (3) coating 200 mu L of bacterial liquid on an LB (lysogeny broth) plate containing kanamycin, performing overnight culture, performing PCR (polymerase chain reaction) sequencing verification, performing liquid culture on positive clones, and extracting plasmids for sequencing verification. Sequencing results show that newly synthesized dcas9 protein gene is inserted into the vector, and the plasmid construction is correct and the bacteria are preserved.

Through web siteshttp://chopchop.cbu.uib.no/sgRNA targeting GFP was designed and sgRNA expression cassettes were ligated to vector 2 by PCR and two IVA's. The gene PCR system is as follows: 5 × TransStart O.RTFastpfu FlyBuffer 10. mu.L, dNTP (2.5mM) 4. mu.L, primer 2. mu.L, TransStart O.RTFastFU Fly DNA Polymerase 1. mu.L, template 1. mu.L, distilled water 32. mu.L, total volume of 50. mu.L. Amplification conditions were pre-denaturation at 95 ℃ for 5 min (1 cycle); denaturation at 95 ℃ for 5 min, annealing at 55 ℃ for 30 sec, extension at 72 ℃ for 30 sec (30 cycles); extension at 72 ℃ for 5 min (1 cycle). The PCR product was digested with DpN1 enzyme at 37 ℃ for 1 hour, 5ul was added to 50. mu.L of DH 5. alpha. competent cells (purchased from Beijing Bomaide Gene technology Co., Ltd.), ice-washed for 30 minutes, heat-shocked at 42 ℃ for 60 seconds, and immediately placed on ice for 2 minutes. Add 250. mu.L SOC medium and shake culture in a shaker at 200rpm and 37 ℃ for 1 hour. And (3) coating 200 mu L of bacterial liquid on an LB (lysogeny broth) plate containing chloramphenicol, performing overnight culture, performing PCR (polymerase chain reaction) sequencing verification, performing liquid culture on positive clones, extracting plasmids, performing sequencing verification, and ensuring correct plasmid construction and bacteria preservation.

The two pellets were transformed into DH 5. alpha. competent cells, ice-cooled for 30 minutes, heat-shocked at 42 ℃ for 90 seconds, and immediately placed on ice for 2 minutes. Add 250. mu.L SOC medium and shake culture in a shaker at 200rpm and 37 ℃ for 1 hour. Since the two plasmids contained kanamycin chloramphenicol and resistance gene, 200. mu.L of the bacterial solution was applied to LB plates containing kanamycin and chloramphenicol and cultured overnight at 37 ℃. Picking single colony every other day and inoculating into 5ml of the culture mediumCulturing in LB liquid culture medium with kanamycin and chloramphenicol at 37 deg.C for 4 hr, adding 1g/L leucine, and detecting GFP expression and OD with 200ul bacterial liquid microplate reader every 2 hr₆₀₀。

Claims

1. A method for regulating gene expression by using a novel dCas9 system is characterized in that the gene expression is more accurately regulated on the translation level through the preference of organisms to codons, the utilization frequency of individual codons and the concentration of tRNA corresponding to the individual codons in a host cell during the intracellular translation process are lower.

2. The method of using dCas9 system to regulate gene expression includes the following steps:

A. the strains used and their codon preference need to be determined;

B. according to the preference of the amino acid codon and the dCas9 gene sequence, the selected amino acid and the rare codon thereof are determined, the codon of the corresponding amino acid in the gene to be regulated is replaced by the corresponding rare codon, and the sequence is artificially synthesized again by using a PCR method;

C. connecting the gene sequence artificially synthesized in the step 2 with a plasmid vector 1, transferring the connecting product into a sensitive cell, selecting a correct single colony through PCR verification, storing a bacterial liquid, and extracting the plasmid 1;

D. designing sgRNA targeting a gene required to be regulated, connecting a sgRNA expression frame to a vector 2 by utilizing PCR (polymerase chain reaction), transferring a connecting product into a sensitive cell, selecting a correct single colony through PCR verification, preserving bacterial liquid, and extracting a plasmid 2;

E. and (3) transforming the two plasmids extracted in the step (3) and the step (4) into the same competent cell, coating a flat plate, selecting a single colony, inoculating the single colony into a culture medium for culture, supplementing corresponding amino acid, and detecting the expression of the regulatory gene.

3. The method of claim 1 or 2, wherein in step (a), the rare codon is any codon that can insert an amino acid into a peptide chain relative to the strain, and the screenable range is applicable to, but not limited to, microorganisms.

4. The method according to claim 1 or 2, wherein in step (B), the rare codons used comprise all the rare codons in a specific species.

5. The method for regulating gene expression using the novel dCas9 system according to claim 1 or 2, wherein in step (E), the amino acid is added in the form of a rare codon contained in the specific microorganism.