RRC ID 33476
Author Martinez-Morales F, Borges AC, Martinez A, Shanmugam KT, Ingram LO.
Title Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction.
Journal J Bacteriol
Abstract A set of vectors which facilitates the sequential integration of new functions into the Escherichia coli chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426-4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single FRT site. The modified vectors contain two FRT sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single FRT site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating Zymomonas mobilis genes encoding the ethanol pathway behind the native chromosomal adhE gene in strains of E. coli K-12 and E. coli B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of E. coli strains with precise deletion of extraneous genes used during construction.
Volume 181(22)
Pages 7143-8
Published 1999-11-1
DOI 10.1128/JB.181.22.7143-7148.1999
PMID 10559184
PMC PMC94193
MeSH Anti-Bacterial Agents / pharmacology Chromosomes, Bacterial / genetics* DNA Nucleotidyltransferases / metabolism Drug Resistance, Microbial / genetics Escherichia coli / drug effects Escherichia coli / enzymology Escherichia coli / genetics* Genetic Markers / genetics* Integrases* Molecular Sequence Data Plasmids / genetics Recombinases Recombination, Genetic* Replicon / genetics* Zymomonas / genetics Zymomonas / metabolism
IF 3.006
Times Cited 68
Prokaryotes E. coli pFT-A pLOI2223 pLOI2224 pLOI2225 pLOI2226 pLOI2227 pLOI2228 pLOI2403