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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
Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli
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.
