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Application of composition gene cluster deletion in reducing drug resistance of escherichia coli to antibiotics

A technology of Escherichia coli and gene clusters, applied in the fields of genetic engineering and biomedicine, can solve problems such as product safety hazards and microbial contamination, and achieve the effects of increased cell volume, high bacterial concentration, and fast growth

Active Publication Date: 2021-07-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Antimicrobial resistance in E. coli leads to microbial contamination and safety concerns for product consumption, study finds

Method used

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  • Application of composition gene cluster deletion in reducing drug resistance of escherichia coli to antibiotics
  • Application of composition gene cluster deletion in reducing drug resistance of escherichia coli to antibiotics
  • Application of composition gene cluster deletion in reducing drug resistance of escherichia coli to antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of engineering bacteria WJW00 (E.coli W3110△gmhD), WJW01, WJW02, WJW03, WJW04, WJW05, WJW06, WJW07 and WJW08

[0054] The construction process of engineering bacteria WJW00 (E.coli W3110△gmhD), WJW01, WJW02, WJW03, WJW04, WJW05, WJW06, WJW07 and WJW08 is recorded in the Chinese invention patent document with publication number CN110387347B; wherein:

[0055]WJW01 strain: Lipopolysaccharide (LPS) includes three parts: lipid A, core sugar and O-antigen, among which lipid A is a highly conserved region, while core sugar and O-antigen are composed of different sugar molecules. The core sugar synthesis gene cluster contains 14 genes, namely waaQ, waaG, waaP, waaS, waaB, waaO, waaR, waaY, waaZ, waaU, waaL, waaC, waaF, gmdD. On the basis of strain WJW00, continue to knock out the remaining 13 genes in the core sugar gene cluster gmdD (gmhD)-waaFC-waaQGPSBORYZUL except the gmhD gene; pick a single colony to verify its sensitivity to ampicillin and kanamy...

Embodiment 2

[0063] Embodiment 2: The drug patch experiment of bacterial strain to multiple drugs

[0064] Specific steps are as follows:

[0065] (1) Escherichia coli W3110, WJW02, WJW04 and WJW07 were respectively inoculated into LB liquid medium, and cultured at 37°C and 200 rpm for 12 hours to prepare seed liquid;

[0066] (2) The prepared seed liquid was measured by initial OD 600 =0.2 Transfer to fresh LB liquid medium, cultivate to logarithmic phase OD at 37°C, 200rpm 600 =1.0, the bacterial suspension was prepared;

[0067] (3) Draw 100 μL of the bacterial suspension obtained in step (2) and spread evenly on the LB solid medium respectively. ) to the corresponding position, and after marking, cultivate in a 37°C incubator for 24 hours, take pictures, and observe the inhibition zone. Among them, Escherichia coli to clindamycin and novobiocin susceptibility test results are shown in Table 2 and figure 1 Shown:

[0068] Table 2: Zones of Inhibition for Clindamycin and Novobiocin...

Embodiment 3

[0071] Embodiment 3: The drug resistance experiment of strain to clindamycin

[0072] 1. Analysis of the resistance of E.coli W3110, WJW02, WJW04 and WJW07 to clindamycin

[0073] (1) In a 96-well plate, add different concentrations of novobiocin to each well by doubling dilution; for each concentration, add 180 μL LB liquid medium plus 20 μL antibiotic stock solution to the first well, fully Mix well, transfer 100 μL to the second well with 100 μL LB, then transfer 100 μL from the second well to the third well with 100 μL LB, and so on.

[0074] The strain was cultured in LB liquid medium overnight to mid-late logarithmic (OD 600 =3.00), the sample was diluted with fresh LB medium, and the bacterial concentration was OD 600 Dilute to 1 x 10 -3 , add 100 μL to the 96-well plate.

[0075] The first well is a blank LB control well that only adds LB medium, no antibiotics and bacterial solution, and the last well is a blank control that does not add antibiotics but only bacte...

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Abstract

The invention discloses an application of composition gene cluster deletion in reducing the drug resistance of escherichia coli to antibiotics, and belongs to the fields of gene engineering and biological medicine. A core sugar gene cluster gmhD-waaQ, an O-antigen gene cluster wbbL-rmlB, an exopolysaccharide gene cluster galF-wza, a 4-type capsular polysaccharide synthetic gene cluster yccC-ymcD and three flagellar gene clusters flhE-motA, fliY-fliR and flgN-flgL of escherichia coli W3110 are knocked out, the strain WJW07 is finally obtained, the MIC of the strain to clindamycin and novobiocin in an LB culture medium is 20 mg / L and 3 mg / L respectively, the MIC of the strain is reduced by 65 times and 250 times compared with that of a wild control strain W3110, and a series of membrane wall structure related synthesis and transport gene clusters are deleted, so that the biomass of the recombinant strain WJW07 is increased, and industrial production and application are facilitated.

Description

technical field [0001] The invention relates to the application of deletion composition gene clusters in reducing the drug resistance of Escherichia coli to antibiotics, and belongs to the fields of genetic engineering and biomedicine. Background technique [0002] Bacterial drug resistance refers to the phenomenon that bacteria are not sensitive to antibacterial drugs, and it is a special form of expression in the process of bacteria's own survival. [0003] Due to the abuse of antibiotics, drug-resistant strains have increased, and they have high resistance to clindamycin, novobiocin and other antibiotics. At present, the resistance of pathogenic strains to antibiotics belongs to acquired resistance. Due to the existence of drug resistance, pathogenic strains are not sensitive to antibiotics, and antibiotics need to be frequently replaced during the treatment of diseases. [0004] Clindamycin belongs to the lincomycin class of antibiotics. As a hydrophobic molecule, it ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12P17/18C12R1/19
CPCC07K14/245C12P17/182Y02A50/30
Inventor 王小元王建莉马文渐梁浩秦晓龙
Owner JIANGNAN UNIV
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