Method for improving drug resistance of escherichia coli and application thereof
A technology of Escherichia coli and drug resistance, which is applied in the fields of genetic engineering and medical engineering, and can solve problems such as product safety hazards and microbial contamination
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0045] Embodiment 1: Construction of knockout plasmid
[0046] Using the CRISPR / Cas9 knockout system to knock out flagella and pilus synthesis and transport gene clusters, four knockout plasmids need to be constructed: pTargetF-flhD-motA, pTargetF-fliY-fliR, pTargetF-flgN-flgL, pTargetF-fimB-fimH , the construction process of the plasmid is as follows:
[0047] (1) Select 20nt N complementary to the target sequence of the target gene 20 sequence, specific N 20 The sequences are the underlined sequences of the primers pTargetF-flhE-motA-F, pTargetF-fliY-fliR-F, pTargetF-flgN-flgL-F, and pTargetF-fimB-fimH-F, and these sequences are modified into the plasmid pTargetF forward primer At the 5' end, forward primers pTargetF-flhD-motA-F, pTargetF-fliY-fliR-F, pTargetF-flgN-flgL-F, and pTargetF-fimB-fimH-F were respectively obtained. Using the plasmid pTargetF as a template, the forward primers pTargetF-flhD-motA-F, pTargetF-fliY-fliR-F, pTargetF-flgN-flgL-F, pTargetF-fimB-fimH-F,...
Embodiment 2
[0054] Embodiment 2: Construction of genetically engineered bacteria WJW010 and WJW011
[0055] The CRISPR / Cas9 knockout system was used to knock out the flagella synthesis and transport gene cluster of wild-type Escherichia coli W3110 to obtain engineering strain WJW010; the pili gene cluster was knocked out to get WJW011. The specific knockout process is as follows:
[0056] (1) Preparation of Escherichia coli electroporation knockout competent
[0057] The plasmid pCas is transformed into Escherichia coli W3110 to obtain recombinant Escherichia coli W3110 / pCas containing the pCas plasmid, and the Escherichia coli W3110 / pCas is activated on the LB solid plate adding 30mg / L Kanamycin (Kan), and inoculated into LB +Kan test tube cultured overnight to obtain seed liquid; transfer the seed liquid to 25mL LB+Kan medium at 1%, and cultivate to OD at 30°C and 200rpm 600 =0.2, add 500 μL L-arabinose solution to induce, continue to culture to OD 600 =0.5, ice bath for 30min; 4°C, ...
Embodiment 3
[0069] Example 3: Drug patch experiment of strains WJW010, WJW011 and wild-type strain W3110
[0070] Specific steps are as follows:
[0071] (1) Escherichia coli W3110 (hereinafter referred to as W3110), flagella gene knockout strain WJW010 and pili knockout strain WJW011 were inoculated into LB liquid medium respectively, and cultured at 37°C and 200rpm for 12h to prepare seed liquid;
[0072] (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;
[0073] (3) Draw 100 μL of the bacterial suspension obtained in step (2) and spread evenly on the LB solid medium respectively. Stick it to the corresponding position, mark it, incubate it in a 37°C incubator for 24 hours, take pictures, and observe the inhibition zone; among them, the results of the cefoxitin drug susceptibility test are shown in Table 3 and figure 1 Shown:
[0074]...
PUM
| Property | Measurement | Unit |
|---|---|---|
| diameter | aaaaa | aaaaa |
| diameter | aaaaa | aaaaa |
| diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More