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Method for Screening Positive Cloned Strains Applied to Anaerobic Bacteria and Facultative Anaerobic Bacteria

A technology of facultative anaerobic bacteria and anaerobic bacteria, applied in the field of genetic engineering, can solve the problems of inability to degrade hydrogen peroxide, inability to destroy superoxide groups, etc., and achieve remarkable effects

Active Publication Date: 2021-02-09
HUAIYIN TEACHERS COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Anaerobes and most facultative anaerobes do not contain catalase to degrade hydrogen peroxide and generally cannot destroy superoxide groups

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Screening Escherichia coli SOD superoxide dismutase target gene in DH5α Bifidobacterium adolescentis Positive clone of ATCC15703

[0018] 1) from Escherichia coli DH5α extracts catalase and SOD genes respectively: Escherichia coli DH5α strain culture, use LB medium, 37 ° C test tube shaking shaker culture to OD660 about 0.6, extract the whole gene of the bacteria and store it in a -20 ° C refrigerator for later use; according to Escherichia coli The heme catalase gene sequence of DH5α was designed with upstream and downstream primers to extract the heme catalase gene and stored in a -20°C refrigerator for later use; according to Escherichia coli The SOD superoxide dismutase gene sequence of DH5α was designed with upstream and downstream primers to extract the SOD catalase gene and stored in a -20°C refrigerator for later use;

[0019] 2) from Bifidobacterium longum ATCC 15707 extracts promoter and terminator genes: Bifidobacterium longum The ATCC...

Embodiment 2

[0023] Example 2: Screening Escherichia coli SOD target gene in DH5α Lacterbaillus acidophilus Positive clone of ATCC4356

[0024] 1) from Escherichia coli DH5α extracts catalase and SOD genes respectively: Escherichia coli DH5α strain culture, use LB medium, 37 ° C test tube shaking shaker culture to OD660 about 0.6, extract the whole gene of the bacteria and store it in a -20 ° C refrigerator for later use; according to Escherichia coli The heme catalase gene sequence of DH5α was designed with upstream and downstream primers to extract the heme catalase gene and stored in a -20°C refrigerator for later use; according to Escherichia coli The SOD superoxide dismutase gene sequence of DH5α was designed with upstream and downstream primers to extract the SOD catalase gene and stored in a -20°C refrigerator for later use;

[0025] 2) from Lacterbaillus acidophilus ATCC4356 extracts promoter and terminator genes: Lacterbaillus acidophilus The ATCC4356 strain was cu...

Embodiment 3

[0029] Example 3: Screening Escherichia coli SOD superoxide dismutase target gene in DH5α Bifidobacterium adolescentis Positive clone of ATCC15703

[0030] 1) from Escherichia coli DH5α extracts catalase and SOD genes respectively: Escherichia coli DH5α strain culture, use LB medium, 37 ° C test tube shaking shaker culture to OD660 about 0.6, extract the whole gene of the bacteria and store it in a -20 ° C refrigerator for later use; according to Escherichia coli The heme catalase gene sequence of DH5α was designed with upstream and downstream primers to extract the heme catalase gene and stored in a -20°C refrigerator for later use; according to Escherichia coli The SOD superoxide dismutase gene sequence of DH5α was designed with upstream and downstream primers to extract the SOD catalase gene and stored in a -20°C refrigerator for later use;

[0031] 2) from Bifidobacterium longum ATCC 15707 extracts promoter and terminator genes: Bifidobacterium longum The ATCC...

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Abstract

The invention discloses a method for screening positive clone strains applied to anaerobic bacteria and facultative anaerobic bacteria. The method for screening positive clone strains comprises the following specific steps: the first step is to Escherichia coli Extract the catalase gene from DH5α; the second step is to extract the promoter and terminator genes from the transformed target bacteria; the third step is to assemble the catalase expression sequence into the target bacteria‑ E. coli Shuttle plasmid; the fourth step carries the plasmid expressing the catalase gene and transforms it into the target bacteria; the fifth step is the verification of the positive clone of the target bacteria; the sixth step is the rapid verification of the positive clone of the target bacteria carrying the target gene. In the present invention, the gene for expressing catalase is carried on the plasmid transformed into anaerobic bacteria or facultative anaerobic bacteria, and H 2 o 2 The phenomenon of bubbles will be generated to judge the positive strain, the method is simple and fast.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for screening positive clone strains applied to anaerobic bacteria and facultative anaerobic bacteria. [0002] technical background [0003] Genetic engineering or recombinant DNA technology refers to the molecular manipulation and construction of genetic information, that is, the isolated or synthesized genes are modified, inserted into vectors, introduced into host cells, and amplified and expressed to obtain a large number of gene products , or cause organisms to exhibit new traits. The term genetic engineering is used to refer to specific genetic manipulation, and it can also refer to the technical system involved in it, the core of which is the technology of constructing recombinant DNA. [0004] In genetic engineering, plasmid transformation into microorganisms is one of the commonly used techniques. The screening of positive clone strains is a nece...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/74C12Q1/04C12R1/01
CPCC12N9/0065C12N15/74C12Q1/04C12Y111/01006
Inventor 贺建龙熊鹏许家兴吴真徐继明
Owner HUAIYIN TEACHERS COLLEGE