title: Construction of a thermo-sensitive pRI857 vector for efficient DNA capturing in Escherichia coli.
abstract: OBJECTIVE
  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 PRpromoter and kanamycin resistance gene (PR-kanR) at 30 °C, but have no effect on PR-kanRgene 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 ~107cfu 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.
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhang
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Huijuan
authors.initials: H
authors.lastname: Su
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Muhan
authors.initials: M
authors.lastname: Yang
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Ge
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Guiyao
authors.initials: G
authors.lastname: Li
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Yi
authors.affiliation: School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, 210094, Jiangsu, China. wangyang@njust.edu.cn.|Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. wangyang@njust.edu.cn.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006358:Hot Temperature; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; D004247:DNA
keywords: CI857 repressor; PR Promoter; Repressor-inactivation; Zero-background cloning