| RRC ID |
33476
|
| 著者 |
Martinez-Morales F, Borges AC, Martinez A, Shanmugam KT, Ingram LO.
|
| タイトル |
Chromosomal integration of heterologous DNA in Escherichia coli with precise removal of markers and replicons used during construction.
|
| ジャーナル |
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.
|
| 巻・号 |
181(22)
|
| ページ |
7143-8
|
| 公開日 |
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
|
| 引用数 |
68
|
|
WOS 分野
|
MICROBIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
pFT-A
pLOI2223
pLOI2224
pLOI2225
pLOI2226
pLOI2227
pLOI2228
pLOI2403 |
