阅读说明：本技术 一种利用新型dCas9调控基因表达的方法 (Method for regulating gene expression by using novel dCas9 ) 是由 霍毅欣 郭丽微 刘玉美 于 2019-09-27 设计创作，主要内容包括：CRISPRi系统由于可以阻断RNA聚合酶复合物的起始或延长来抑制基因转录,为了可控地调节基因的转录,dCas9基因会与各种诱导型启动子连接后进行表达。然而由于一些诱导型启动子宽松的调控机制,在不添加诱导剂的情况下,dCas9仍具有微弱的表达能力,进而对目的基因进行部分抑制。工业生产的大规模发酵中需要实现基因的精准调控,故诱导型启动子引起的泄露表达问题亟待解决。本发明的目的在于提供一种利用新型dCas9系统调控基因表达的方法,利用密码子偏好性和tRNA氨酰化水平的差异,在翻译水平上,简单有效地通过补加氨基酸实现dCas9基因的表达调控。将dCas9基因中的氨基酸替换为其稀有密码子形式,外源添加对应氨基酸,可以使其恢复表达有功能的蛋白而发挥抑制作用。(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.)

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.

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.