[Gene knockout and knockin on the Escherichia coli lac operon loci using pBR322-red system].
pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
Chen
Wei
W
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
Yu
Mei
M
Li
Shan-Hu
SH
Wang
Ming-Gang
MG
Zhou
Jian-Guang
JG
D010582:Bacteriophage lambda / Q000235:genetics*; D002876:Chromosomes, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D055879:Gene Knock-In Techniques / Q000379:methods*; D055786:Gene Knockout Techniques / Q000379:methods*; D006801:Humans; D007763:Lac Operon / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic