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Citrobacter welchii TpiA gene knockout mutant strain and application thereof

A Citrobacter, gene knockout technology, applied in the field of genetic engineering, can solve problems such as difficult to remove fungicides and biofilms

Active Publication Date: 2022-01-18
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The removal of wound bacterial biofilms has long been a major problem, and 80% of clinical infections are related to biofilms. Bacteria are resistant to antibiotics because they are prone to biofilms, and general fungicides are difficult to remove

Method used

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  • Citrobacter welchii TpiA gene knockout mutant strain and application thereof
  • Citrobacter welchii TpiA gene knockout mutant strain and application thereof
  • Citrobacter welchii TpiA gene knockout mutant strain and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Construction of Citrobacter welmannii tpiA gene knockout mutant

[0040] 1. Construction of tpiA knockout vector

[0041]First, the upstream homologous sequence (963bp; its nucleotide sequence is shown in SEQ ID NO.1) of the tpiA gene (768bp; its nucleotide sequence is shown in SEQ ID NO. 2) and downstream sequence (795bp; its nucleotide sequence is shown in SEQ ID NO.3), and the pYG4 plasmid sequence (5796bp; its nucleotide sequence is shown in SEQ ID NO.4), copied to the software Relevant positions of the multi-fragment clone (ClonExpress MultiS) of CE Design V1.04, and related settings: vector linearization method selects single enzyme digestion For linearization, select 2 for the number of inserts, and select BglII for the linearization restriction site. The tpiA-up-F / tpiA-up-R and tpiA-down-F / tpiA-down-R primer pairs designed and output by software CE Design V1.04 were entrusted to Guangzhou Branch of Beijing Qingke Xinye Biotechnology Co., Ltd. Primer...

Embodiment 2

[0064] Example 2 Determination of biofilm formation ability of tpiA knockout

[0065] To measure the biofilm formation ability of ΔtpiA, the experimental steps are mainly: use LB to culture ΔtpiA and wild Citrobacter weimanii GDFMZ BF-8 overnight, and adjust the concentration of the bacterial solution to OD with fresh LB the next day 600 =0.5 for use; add 360 μl of fresh sterile LB culture medium to 48-well plates (Greiner bio-one), and then add 40 μl of bacteria solution with adjusted bacterial concentration; Add 0, 64, 128, 256 μg / mL concentration of imatinib mesylate (McLean, CAS: 220127-57-1) and put it in a 30°C incubator for 1 day, discard the planktonic bacteria first, and Wash the 48-well plate, and then stain it with 0.1% crystal violet. After washing off the excess dye with sterilized water, wash off the crystal violet remaining on the wall of the 48-well plate with 95% alcohol, and use enzyme labeling The light absorption value of the sample at 590nm is measured by...

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Abstract

The invention discloses a citrobacter welchii tpiA gene knockout mutant strain and application thereof, and belongs to the field of gene engineering. The citrobacter welchii tpiA gene knockout mutant strain is obtained by completely knocking out coding genes from the first site to the 768th site of a citrobacter welchii wild strain tpiA gene, and the coding gene sequence from the first site to the 768th site of the tpiA gene is as shown in SEQ ID NO.1. The citrobacter welchii GDFMZ BF-8 delta tpiA has a preservation number of GDMCC 61942, and experiments prove that the mutant strain has the capability of reducing the biofilm formation amount of the citrobacter welchii. Meanwhile, the invention further provides a method for reducing the biofilm formation amount of the citrobacter welchii. The invention also proves that the tpiA gene is an action site of imatinib mesylate and can be used in a target site controlled by a biofilm.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a gene knockout mutant strain of Citrobacter wilsonii with reduced biofilm formation and application thereof. Background technique [0002] Bacteria of the genus Citrobacter, nutrient energy organic nutrition, can use citrate as the only carbon source for growth, bacteria of this genus are all Gram-negative bacilli, usually have perinatal flagella, facultative anaerobic, and have respiratory and Fermentation Two types of metabolism, fermentation of glucose to produce acid and gas; bacteria of this genus are often found in human and animal feces, may be normal intestinal resident bacteria, and are also found in soil, water, sewage and food, but It is also frequently isolated from clinical samples as an opportunistic pathogen. Citrobacter was detected in the pathogenic bacteria isolated from the feces of patients with bacterial diarrhea, and caused patients with varying degrees ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/61A61K45/00A61K31/506A61P31/04C12R1/01
CPCC12N9/90A61K45/00A61K31/506A61P31/04C12Y503/01001Y02A50/30
Inventor 王颖思周刚谢小保彭红施庆珊李彩玲李素娟彭如群
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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