Convenient and reversible site-specific targeting of exogenous DNA into a bacterial chromosome by use of the FLP recombinase: the FLIRT system.
We have created a system that utilizes the FLP recombinase of yeast to introduce exogenous cloned DNA reversibly at defined locations in the Escherichia coli chromosome. Recombination target (FRT) sites can be introduced permanently at random locations in the chromosome on a modified Tn5 transposon, now designed so that the inserted FRT can be detected and its location mapped with base pair resolution. FLP recombinase is provided as needed through the regulated expression of its gene on a plasmid. Exogenous DNA is introduced on a cloning vector that contains an FRT, selectable markers, and a replication origin designed to be deleted prior to electroporation for targeting purposes. High yields of targeted integrants are obtained, even in a recA background. This system permits rapid and precise excision of the introduced DNA when needed, without destroying the cells. The efficiency of targeting appears to be affected only modestly by transcription initiation upstream of the chromosomal FRT site. With rare exceptions, FRTs introduced to the bacterial chromosome are targeted with high efficiency regardless of their location. The system should facilitate studies of bacterial genome structure and function, simplify a wide range of chromosomal cloning applications, and generally enhance the utility of E. coli as an experimental organism in biotechnology.
Huang
L C
LC
Department of Biochemistry, University of Wisconsin-Madison, 53706, USA.
Wood
E A
EA
Cox
M M
MM
D015139:Blotting, Southern; D002876:Chromosomes, Bacterial / Q000235:genetics*; D004247:DNA / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005819:Genetic Markers; D005822:Genetic Vectors; D016680:Genome, Bacterial; D007763:Lac Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D004251:DNA Transposable Elements; D005819:Genetic Markers; D004247:DNA; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase