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Journal of Bacteriology, November 2001, p. 6384-6393, Vol. 183, No. 21
Department of Biological Sciences, Purdue
University, West Lafayette, Indiana 47907
Received 15 June 2001/Accepted 18 August 2001
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.
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.21.6384-6393.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Conditional-Replication, Integration, Excision, and
Retrieval Plasmid-Host Systems for Gene Structure-Function Studies
of Bacteria
and
*
Corresponding author. Mailing address: Department of
Biological Sciences, Purdue University, West Lafayette, IN 47907. Phone: (765) 494-8034. Fax: (765) 494-0876. E-mail:
BLW{at}bilbo.bio.purdue.edu.
Present address: ARPIDA, 4142 Münchenstein, Switzerland.
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