Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli
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Updated: Mar 6, 2026
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Kai Zhang1, Huijuan Su1, Muhan Yang1
1School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
Objectives:
To establish a positive cloning system with a zero background for high-throughput DNA cloning purpose.
Results:
The cloning vector, pRI857, and the genomic-library construction vector, pRI857-BAC, were constructed based on the mechanism of expression of the thermo-sensitive cI857 repressor gene that can stringently repress the PR promoter and kanamycin resistance gene (PR-kan R ) at 30 °C, but have no effect on PR-kan R gene at 37 °C or at higher temperatures. When the pRI857 vectors were transformed into E. coli with or without a target foreign DNA fragment inserted at the BfrBI site of the cI857 gene, only colonies with the foreign DNA fragment survive. We extended this method to construct a pRI857-BAC vector for genomic library cloning which displays an efficiency of ~107 cfu per µg of genomic DNA, with no empty vectors detected.
Conclusions:
Cloning by indirect activation of resistance marker gene represents a novel DNA-capturing system, which can be widely applied for high-throughput DNA cloning.
