[Development of a new recombineering system by gap repair].
Using lambda phage Red recombinase mediated in vivo homologous recombination system, a 6.7 kb lambda PL operon sequence including the Red encoding genes was subcloned into pBR322 by gap repair technique, and generated a pBR322-Red recombinant plasmid that can provide the Red recombination function and can be transferred into many kinds of bacteria. To confirm the recombination functions of pBR322-Red, a single-stranded 70-bases oligo was introduced into W3110 by electroporation to create a single base T-->G mutation in galK gene on the bacterial chromosome. The result demonstrated that a new lambda Red-mediated recombineering system based on pBR322-Red was successfully established.
Li
Shan-Hu
SH
Institute of Biotechnology, Academy of Military Medical Sciences, Beijing 100850, China.
Hong
Xin
X
Yu
Mei
M
Chen
Wei
W
Huang
Cui-Fen
CF
Zhou
Jian-Guang
JG
D010582:Bacteriophage lambda / Q000201:enzymology / Q000235:genetics; D004260:DNA Repair / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D009876:Operon; D010957:Plasmids; D017354:Point Mutation; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D045522:Recombinases; D005686:Galactokinase