CN110592126A

CN110592126A - Method for regulating gene expression at translation level by using rare codon

Info

Publication number: CN110592126A
Application number: CN201910923897.8A
Authority: CN
Inventors: 霍毅欣; 郑博; 王宁
Original assignee: Beijing University of Technology
Current assignee: Beijing University of Technology; Beijing Institute of Technology BIT
Priority date: 2019-09-27
Filing date: 2019-09-27
Publication date: 2019-12-20

Abstract

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.

Description

Method for regulating gene expression at translation level by using rare codon

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.

Example 1 method for controlling GFP expression at the translational level Using rare codons

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 the green fluorescent protein GFP are replaced by rare codons CUA, and a green fluorescent protein gene shown in a sequence 1 is obtained by adopting a gene total synthesis method.

The gene total synthesis system is as follows:fastpfu Fly Buffer 10. mu.L, dNTP (2.5mM) 4. mu.L, primer premix 2. mu.L,FastPfu Fly DNA Polymerase 1. mu.L, distilled water 35. mu.L, total volume 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 contains the green fluorescent protein gene and 200 bases respectively at the upstream and downstream thereof, and is connected to a pGFP vector. A connection system: 1.5. mu.L of PCR amplification product, 1. mu.L of pGFP vector 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 plate containing chloramphenicol, performing overnight culture, performing PCR sequencing verification, performing liquid culture on positive clones, and extracting plasmids for sequencing verification. Sequencing results show that newly synthesized green is inserted into the vectorThe chromo-fluorescent protein and 200 basic groups on the upstream and the downstream thereof prove that the plasmid is constructed correctly and the bacteria is preserved.

Coating the preserved strain with a chloramphenicol resistant plate, overnight culturing at 37 deg.C, selecting single colony every other day, inoculating into 5ml LB liquid medium containing chloramphenicol, culturing at 37 deg.C for 4 hr, supplementing 1g/L leucine, and detecting GFP expression and OD with 200ul bacterial liquid enzyme labeling instrument every 2 hr₆₀₀。

Claims

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.