Fluorescent plasmid pTD103luxl-sfGFP-apr, fluorescent strain, construction method and application

By replacing the resistance gene in the fluorescent plasmid, a fluorescent plasmid pTD103luxl-sfGFP-apr suitable for Klebsiella pneumoniae was constructed, which solved the problem of resistance of the bacteria to commonly used fluorescent plasmids and achieved efficient fluorescent labeling and research.

CN119685362APending Publication Date: 2025-03-25XINXIANG MEDICAL UNIV
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Patent Information

Application Number
CN202411902264.6
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Filing Date
2024-12-23
Publication Date
2025-03-25

AI Technical Summary

Technical Problem

Existing research methods are difficult to effectively study the adhesion process of Klebsiella pneumoniae, especially because Klebsiella pneumoniae is naturally resistant to ampicillin, which leads to the common fluorescent plasmids not being used in this bacteria.

Method used

By designing primers containing overlapping fragments with the plasmid, using seamless cloning technology to replace the ampicillin resistance gene of the luminescent plasmid itself with the aberramycin resistance gene, a fluorescent plasmid pTD103luxl-sfGFP-apr suitable for Klebsiella pneumoniae was constructed.

Benefits of technology

It realizes efficient expression of fluorescent plasmids in Klebsiella pneumoniae, simplifies experimental steps, reduces operation difficulty, and improves the intuitiveness and accuracy of experimental results.

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Abstract

The invention discloses a fluorescent plasmid pTD103luxl-sfGFP-apr as well as a construction method and application of a fluorescent strain, and relates to the technical field of biochemistry. The specific method comprises the following steps: amplifying an Apr gene by taking Apr-fragment-ptd-F and Apr-fragment-ptd-R as primers, and carrying out amplification on the Apr gene by taking the Apr-fragment-ptd-F and the Apr-fragment-ptd-R as the primers; the method comprises the following steps: carrying out reverse amplification on a pTD103luxl-sfGFP carrier by taking ptd103-vector-F and ptd103-vector-R as primers, and carrying out reverse amplification on the pTD103luxl-sfGFP carrier by taking ptd103-vector-R as primers; carrying out a recombination reaction on the gel recovered Apr gene segment and the reverse amplification segment of the pTD103luxl-sfGFP vector, so as to obtain a recombination product; and adding the recombinant product into competent cells to react, and then coating the competent cells with the recombinant product in an LB culture medium containing Apr resistance to extract plasmids, namely the fluorescent plasmid pTD103luxl-sfGFP-apr. The fluorescent plasmid constructed by the invention is simple in steps, low in material consumption and high in conversion efficiency, can be expressed in various klebsiella pneumoniae, and can be used for colonization of the klebsiella pneumoniae.
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