RRC ID 33477
Author Haldimann A, Wanner BL.
Title Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.
Journal 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.
Volume 183(21)
Pages 6384-93
Published 2001-11
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 3.219
Times Cited 307
WOS Category MICROBIOLOGY
Resource
Prokaryotes E. coli 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