| RRC ID |
33477
|
| 著者 |
Haldimann A, Wanner BL.
|
| タイトル |
Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.
|
| ジャーナル |
J Bacteriol
|
| Abstract |
We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision ("curing"), or retrieval of the CRIM plasmids.
|
| 巻・号 |
183(21)
|
| ページ |
6384-93
|
| 公開日 |
2001-11-1
|
| DOI |
10.1128/JB.183.21.6384-6393.2001
|
| PMID |
11591683
|
| PMC |
PMC100134
|
| MeSH |
Attachment Sites, Microbiological
DNA Nucleotidyltransferases / genetics
DNA Nucleotidyltransferases / physiology
DNA Replication
DNA, Bacterial / genetics
Escherichia coli / genetics*
Gene Dosage
Gene Library*
Genes, Bacterial*
Genetic Vectors*
Integrases / genetics
Integrases / physiology
Molecular Sequence Data
Plasmids*
Recombination, Genetic*
Structure-Activity Relationship
Transformation, Bacterial
Viral Proteins*
|
| IF |
3.006
|
| 引用数 |
381
|
|
WOS 分野
|
MICROBIOLOGY
|
| リソース情報 |
| 原核生物(大腸菌) |
pAH120
pAH121
pAH122
pAH123
pAH125
pAH129
pAH130
pAH131
pAH143
pAH144
pAH145
pAH150
pAH152
pAH153
pAH154
pAH156
pAH162
pAH55
pAH56
pAH57
pAH63
pAH68
pAH69
pAH70
pAH81
pAH83
pAH95
pCAH56
pCAH63
pINT-ts
pLA2 |
