阅读说明：本技术 一种利用稀有密码子在翻译水平调控基因表达的方法 (Method for regulating gene expression at translation level by using rare codon ) 是由 霍毅欣 郑博 王宁 于 2019-09-27 设计创作，主要内容包括：转录水平的基因表达调控需要严谨调控的启动子,同时要求启动子具有可诱导、可放大。现有基因表达调控系统诱导剂价格昂贵且需要特定培养基成分很难用于大规模发酵。将代谢途径基因中的氨基酸密码子替换成其稀有形式,可通过是否补加相应氨基酸实现该基因表达调控,诱导物相对廉价易得对细胞无毒害作用,可实现基因翻译水平更加精准的调控,减少泄露表达,且不会干扰到其他基因的表达和影响菌体的正常生长。(The regulation of gene expression at the transcriptional level requires a strictly regulated promoter, while the promoter is required to be inducible and amplifiable. The existing gene expression regulation system inducer is expensive and needs specific culture medium components which are difficult to be used for large-scale fermentation. The amino acid codon in the metabolic pathway gene is replaced into a rare form, the gene expression regulation can be realized by whether corresponding amino acid is supplemented or not, the inducer is relatively cheap and easily available, has no toxic or harmful effect on cells, can realize more accurate regulation of gene translation level, reduces leakage expression, and does not interfere the expression of other genes and influence the normal growth of thalli.)

1. A method for regulating gene expression by using rare codon at translation level is characterized by that it utilizes the bias of organism to codon, and in the course of intracellular translation its individual codon utilization frequency and its correspondent tRNA concentration are lower in host cell, and can change the amino acid codon in the metabolic pathway gene into its rare form so as to implement gene expression regulation at translation level.

2. The method of using rare codons to regulate gene expression at the translational level according to the claim, characterized by the following steps:

A. it is necessary to determine the strain used and the gene to be regulated and to determine the codon preference of the strain used.

B. According to the preference of the amino acid codon and the sequence of the regulatory gene, 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, transferring the connecting product into a sensitive cell, selecting a correct single colony through PCR verification, storing a bacterial liquid, and extracting a plasmid;

D. coating the strain containing the correct plasmid obtained in the step 3 on a flat plate for culturing;

E. selecting single colony, inoculating to culture medium, adding corresponding amino acid, and detecting expression amount of regulatory gene and thallus concentration.

3. The method for regulating gene expression at the translational level using rare codons as set forth in claim 1 or 2, wherein the rare codons are any codons that can insert amino acids into peptide chains with respect to strains in the step (A) within a range applicable to all microorganisms.

4. The method for regulating gene expression at the translational level using rare codons as set forth in claim 1 or 2, wherein in the step (B), the synthetic genes include metabolic pathway genes of respective organisms, essential genes of microorganisms.

5. The method for regulating gene expression at the translational level using rare codon according to claim 1 or 2, wherein in the step (E), the amino acid to be added is an amino acid having the form of rare codon in the specific microorganism.

Technical Field

The invention relates to a method for regulating gene expression at translation level by using rare codons, belonging to the technical field of biological engineering.

Background art:

the regulation of gene expression at the transcriptional level requires a strictly regulated promoter, while the promoter is required to be inducible and amplifiable. The existing gene expression regulation system inducer is expensive and needs specific culture medium components which are difficult to be used for large-scale fermentation.

The amino acid codon in the metabolic pathway gene is replaced by a rare form, the gene is not expressed under the condition of amino acid deficiency, and the gene expression can be realized by externally supplementing corresponding amino acid. The use of rare codons for gene expression regulation has a number of advantages. Firstly, more accurate regulation and control of gene translation level is realized, and leakage expression is reduced. Secondly, the regulation and control of the gene expression by the rare codon only acts on the gene containing the codon, and the expression of other genes can not be interfered and the normal growth of thalli can not be influenced; the inducer is amino acid which is relatively cheap and easily obtained and has no toxic effect on cells.

Disclosure of Invention

The invention aims to provide a method for regulating and controlling gene expression at a translation level by using rare codons, which realizes gene expression regulation and control simply and effectively by supplementing amino acid at the translation level by using the codon preference and the difference of tRNA aminoacylation level and provides a new method for regulating and controlling gene expression of metabolic engineering.

According to the technical scheme provided by the invention, the method for regulating and controlling gene expression at the translation level by using rare codons adopts the following method steps:

1. it is necessary to determine the strain used and the gene to be regulated and to determine the codon preference of the strain used.

2. According to the preference of amino acid codon and the sequence of the regulatory gene, 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.

3. Connecting the gene sequence artificially synthesized in the step 2 with a plasmid vector, transferring the connecting product into escherichia coli, selecting a correct single colony through PCR verification, preserving bacterial liquid, and extracting plasmids.

4. The strain containing the correct plasmid obtained in step 3 is spread on a plate and cultured.

5. Selecting single colony, inoculating to culture medium, adding corresponding amino acid, and detecting expression amount of regulatory gene and thallus concentration.

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.