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Mutant for improving gene expression quantity of purine operon pur and construction method and application thereof

A technology of gene expression quantity and construction method, applied in the directions of microorganism-based methods, biochemical equipment and methods, resistance to vector-borne diseases, etc., to achieve the effect of increasing yield and improving gene expression level

Active Publication Date: 2021-06-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The role of the DNA-binding transcriptional two-component regulator ArcA in regulating the expression of the purine operon pur gene and its application in purine biosynthesis have not been reported yet

Method used

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  • Mutant for improving gene expression quantity of purine operon pur and construction method and application thereof
  • Mutant for improving gene expression quantity of purine operon pur and construction method and application thereof
  • Mutant for improving gene expression quantity of purine operon pur and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of a mutant E. coliW3110ΔarcA that increases the expression of the purine operon pur gene.

[0054] The P1 phage transduction method was used to knock out the DNA-binding transcriptional two-component regulator arcA (Gene ID of arcA: 948874) in the genome of Escherichia coli E. coliW3110, and a mutant strain E. coliW3110△arcA was constructed. Specific steps are as follows:

[0055] 1) Preparation of donor strain cleavage library

[0056] Add 4ml of heated and melted 0.4% agar medium to a 10ml sterile EP tube, add 400 μL of overnight cultured donor strain JW4364 (the donor strain is derived from the Keio Collection library, which can be purchased commercially, and the KeioCollection library construction method is as follows: BabaT, et al. Construction of Escherichia coli K-12in-frame, single-gene knockout mutants: the Keio collection. Molecular Systems Biology 2006, 2 (1): 1-11 "described in), add 10 μ L of stored phage stock solution, Mix the s...

Embodiment 2

[0061] Example 2. Detection of the expression level of the purine operon pur gene.

[0062] In this example, the quantitative PCR technique was used to detect the relative gene expression levels of the purine operon pur of the mutant strain E.coli W3110ΔarcA constructed in Example 1 and the wild strain E.coli W3110. Specific steps are as follows;

[0063] 1) Extract the total RNA of mutant strain E.coliW3110△arcA and wild strain E.coli W3110 by using EASYspin Plus Adelaide Bacterial RNA Rapid Extraction Kit;

[0064] 2) respectively detect the 260nm absorbance value of the total RNA extracted by the wild strain and the mutant strain, and determine the concentration of the RNA;

[0065] 3) After adjusting the RNA concentration to be consistent, use gDNAErase in the PrimerScriptTMRT reagentKit kit to remove the remaining genomic DNA in the RNA, and perform reverse transcription into cDNA;

[0066] 4) The constitutively expressed gene rpoD was used as an internal reference, and...

Embodiment 3

[0068] Example 3. Construction of a hypoxanthine synthetic strain.

[0069] 1) Preparation of mutated PRPP transamidase gene purF:

[0070] Using the genomic DNA of Escherichia coli as a template, using primers SEQ ID NO.4 and SEQ ID NO.6 to clone the upstream fragment of the K326Q mutated PRPP transamidase gene purF, using primers SEQ ID NO.5 and SEQ ID NO.7 The downstream fragment of the PRPP transamidase gene purF obtained by cloning the K326Q mutation, using the fragment bridging method to bridge the upstream and downstream fragments of the PRPP transamidase gene purF of the K326Q mutation with primers SEQ ID NO.4 and SEQ ID NO.5, obtains such as K326Q mutated PRPP transamidase gene purF shown in SEQ ID NO.3;

[0071] With the K326Q mutated PRPP transamidase gene purF as a template, the upstream fragment of the P410W mutated PRPP transamidase gene purF was cloned with primers SEQ ID NO.4 and SEQ ID NO.8, and with primers SEQ ID NO.5 and SEQ ID NO .9 Cloning obtains the d...

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Abstract

The invention discloses a mutant for improving the gene expression quantity of a purine operon pur and a construction method and application thereof, and belongs to the technical field of gene engineering. Aiming at the problems of transcription inhibition of a purine operon pur and difficulty in accumulation of a purine compound in a purine de novo synthesis route, the mutant for improving the gene expression quantity of the purine operon pur is provided, and the mutant is constructed by knocking out DNA in escherichia coli and combining with a transcription two-component regulatory factor arcA (Genbank accession number is 948874). The mutant is further used as a starting strain, a mutated PRPP synthetase gene prs and a mutated PRPP transamidase gene purF are subjected to overexpression, and a recombinant strain with high yield of hypoxanthine is constructed. According to the mutant, regulation of the regulatory factor arcA in gene expression of the purine operon pur and application of the regulatory factor arcA in purine biosynthesis are realized for the first time.

Description

technical field [0001] The invention relates to a mutant for increasing the expression of purine operon pur gene, its construction method and application, and belongs to the technical field of genetic engineering. Background technique [0002] Purine compounds are important life-active substances in cells, and play important roles in the synthesis and transmission of genetic material, signal transduction, and energy supply in cells. In addition, purine compounds can be used as precursors to synthesize a variety of drugs, and are also widely used in the diagnosis and treatment of diseases and life science research. For example, the early mercaptopurine drugs, and the late fludarabine, clorabine and other purine nucleoside drugs play an important role in cancer treatment. [0003] The de novo synthesis route of purine has long been proven. The de novo synthesis of purine mainly uses glutamine and 5-phosphoribosyl pyrophosphate (PRPP) as precursors. After 10 steps of reaction,...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P19/32C12R1/19
CPCC07K14/245C12P19/32Y02A50/30
Inventor 赵广刘敏咸漠富盈昕
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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