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PMID: 11591683 Published · ppublish English Evaluation Study Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.

Journal of bacteriology ·Vol. 183 ·No. 21 ·2001-11-00 ·Pages 6384-93

Haldimann A, Wanner BL

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.

MeSH Terms
Attachment Sites, Microbiological DNA Nucleotidyltransferases/genetics,physiology DNA Replication DNA, Bacterial/genetics Escherichia coli/genetics Gene Dosage Gene Library Genes, Bacterial Genetic Vectors Integrases/genetics,physiology Molecular Sequence Data Plasmids Recombination, Genetic Structure-Activity Relationship Transformation, Bacterial Viral Proteins
Chemicals
DNA, Bacterial Viral Proteins DNA Nucleotidyltransferases Integrases excisionase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Haldimann A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA.
Wanner B L
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Article Info
Journal
Journal of bacteriology
Abbr.
J Bacteriol
ISSN
0021-9193
DOI
10.1128/JB.183.21.6384-6393.2001
Published
2001-11-00
Pages
6384-93
Language
English
Region
United States
NLM ID
2985120R
PMCID
PMC100134
Subset
IM
Grants
NIAID NIH HHS · F33 AI010093 · United States
NIAID NIH HHS · AI8045 · United States
NIAID NIH HHS · F33AI10093 · United States
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External Links
PubMed source DOI: 10.1128/JB.183.21.6384-6393.2001 PMC Full Text View journal details
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