Construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae

A Klebsiella pneumoniae, no resistance marker technology, applied in the field of genetic engineering, to achieve the effect of high luminous intensity, strong stability, easy and quick operation

Pending Publication Date: 2021-02-09
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of the Xer-cise system to excise exogenous resistance genes has only been reported in recent years, and it has been used for the genetic manipulation of Mycobacterium, Acinetobacter baumannii and Pseudomonas aeruginosa, etc. Kp

Method used

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  • Construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae
  • Construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae
  • Construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae

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specific Embodiment approach

[0058] The present invention provides a method for constructing self-luminescent Klebsiella pneumoniae without resistance markers, said Klebsiella pneumoniae is constructed by transfer plasmid pTXR and helper plasmid pUCTns; said construction method comprises the following steps:

[0059] S1. Prepare to transfer plasmid pTXR and helper plasmid pUCTns;

[0060] S2. Transfer the helper plasmid pUCTns expressing the transposase gene into Klebsiella pneumoniae (Kp) competent cells, and apply it to Kanamycin (Kanamycin, referred to as KAN) resistant LB medium (Luria-Bertani Culture medium, be called for short LB) plate, obtain the Klebsiella pneumoniae containing helper plasmid pUCTns;

[0061] S3. Prepare the competent Klebsiella pneumoniae containing the auxiliary plasmid pUCTns, then transfer the transfer plasmid pTXR into the Klebsiella pneumoniae competent cells containing the auxiliary plasmid pUCTns, and apply apramycin (APR) anti- Active LB culture medium plate, obtain the...

Embodiment 1

[0077] Embodiment 1, construction of transfer plasmid pTXR and helper plasmid pUCTns:

[0078] (1) According to the process figure 1 Construct the transfer plasmid pTXR, such as figure 1 As shown, the plasmid pTXR contains AMP resistance gene (bla), replication origin (ori R6kγ), APR resistance gene (apr), luxCDABE gene, direct repeat sequence difR and difL; the functions of each component are as follows:

[0079] luxCDABE: the gene related to autonomous luminescence, which is the enzyme gene required for luminescence, and the expression of this gene enables the host bacteria to emit luminescence autonomously;

[0080] APR resistance gene (apr): apr is a resistance selection marker used for screening to obtain the target strain; after apr is expressed in Kp and E. Growth medium; APR is a resistance screening drug, the APR concentration for Kp is 30 μg / mL, and the APR concentration for Escherichia coli is 50 μg / mL;

[0081] Direct repeat sequence dif: This sequence can be r...

Embodiment 2

[0106] Embodiment 2, a kind of construction method of SfAlKp:

[0107] 1. Electroconversion

[0108] (1) Preparation of Kp competent cells: Take out the frozen Kp from the -80°C refrigerator, put it on crushed ice to thaw, connect it to LB solid medium by streaking method, and culture it overnight. Pick a single colony and transfer it to 10 mL of LB liquid medium, and cultivate it on a shaker at 37°C. OD 600 When it is 0.3-0.8, immediately put it on ice for 30 minutes, centrifuge at 4°C, 4000rpm for 5min, remove the supernatant, add 10mL of pre-cooled sterilized double-distilled water to resuspend the bacteria, repeat washing once, and then centrifuge at 4°C, 4000rpm for 5min , remove the supernatant, add 400 μL of pre-cooled sterilized double-distilled water to resuspend the bacteria, aliquot into 100 μL / tube, and store in a -80°C refrigerator;

[0109] (2) 100 μL of Kp competent cells and 10 μL (concentration of about 300 ng / μL) of plasmid pUCTns were mixed and transferre...

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Abstract

The invention relates to the technical field of gene engineering, in particular to a construction method and application of resistance-marker-free self-luminous klebsiella pneumoniae. The klebsiella pneumoniae is constructed from transfer plasmids pTXR and auxiliary plasmids pUCTns. The method comprises the following steps: S1, preparing the transfer plasmids and the auxiliary plasmids; S2, transferring the auxiliary plasmids for expressing transposase genes into klebsiella pneumoniae competent cells, and coating a kanamycin-resistant LB culture medium plate with the auxiliary plasmids; and S3, preparing the klebsiella pneumoniae containing the auxiliary plasmids into a competent state, then transferring the transfer plasmids into the klebsiella pneumoniae competent cells containing the auxiliary plasmids, and coating an APR-resistant LB culture medium plate with the klebsiella pneumoniae competent cells containing the auxiliary plasmids to obtain the self-luminous klebsiella pneumoniae. According to the construction method disclosed by the invention, exogenous dissociation enzyme does not need to be artificially expressed, the operation is simple, convenient and rapid, and the constructed SfAlKp can emit strong light without adding any substrate; and moreover, the SfAlKp has no resistance genes, so that potential side effects on screening and evaluation of drugs due to cross drug resistance caused by the resistance genes can be avoided.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application of self-luminescent Klebsiella pneumoniae without resistance markers. Background technique [0002] Kp is a Gram-negative bacillus, which is an important conditional pathogen, often causing community-acquired infection and nosocomial infection; when the patient's immunity is weakened due to long-term hospitalization, surgery, catheter indwelling, etc., or long-term heavy use of antibiotics leads to Infection can be caused when the flora is out of balance, and infection can also be caused by contact between patients, medical staff and patients, medical equipment, etc.; 14%-20% of hospital urinary system infections, respiratory infections and sepsis are caused by Kp. In recent years, Kp has become an important conditional pathogen second only to Escherichia coli. Usually, aminoglycoside antibiotics are the first choice for the treat...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/65C12N15/66C12N1/21C12R1/22
CPCC12N15/63C12N15/65C12N15/66C12N1/20C07K14/26
Inventor 张天宇田茜溶高亚敏王帅余崴方翠婷张静然
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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