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

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 ~10(7) 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.