This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cronan, J. E. Search for Related Content PubMed PubMed Citation Articles by Cronan, J. E.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2003, p. 6522-6529, Vol. 185, No. 22
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.22.6522-6529.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cosmid-Based System for Transient Expression and Absolute Off-to-On Transcriptional Control of Escherichia coli Genes

Departments of Microbiology and Biochemistry, University of Illinois, Urbana, Illinois 61801

Received 24 June 2003/ Accepted 12 August 2003

Cosmids are plasmids that contain the phage sequences (cos) required for packaging of the phage DNA into the virion. Induction of a prophage in an Escherichia coli strain carrying a cosmid results in lysates containing phage particles that are filled with cosmid DNA. However, the lysates also contain a large excess of infectious phage particles which complicate use of the packaged cosmids. I report that cosmids packaged by induction of a strain carrying a prophage with an altered cos region results in lysates containing very high levels (>10¹⁰/ml) of particles that contain cosmid DNA together with very few infectious phage particles. These lysates can be used to transduce cosmid DNA into all of the cells of a growing culture with minimal physiological disturbance. When the cosmid carries a conditionally active origin of replication, transductional introduction of the cosmid under nonreplicative conditions provides a system of transient expression. Transient expression has been used to make a recA strain temporarily recombination proficient and to temporarily introduce a site-specific recombinase. Transductional introduction of a cosmid also allows absolute off-to-on transcriptional control of nonessential genes. Two examples are given showing that when a strain carrying a null mutation in the gene of interest is transduced with a packaged cosmid carrying a functional copy of that gene, the expression of the gene rapidly goes from absolutely off to high-level expression. Additional possible uses of in vivo-packaged cosmids are proposed.

This article has been cited by other articles:

• Ting, H., Kouzminova, E. A., Kuzminov, A. (2008). Synthetic Lethality with the dut Defect in Escherichia coli Reveals Layers of DNA Damage of Increasing Complexity Due to Uracil Incorporation. J. Bacteriol. 190: 5841-5854 [Abstract] [Full Text]  
• De Lay, N. R., Cronan, J. E. (2008). Genetic Interaction Between the Escherichia coli AcpT Phosphopantetheinyl Transferase and the YejM Inner Membrane Protein. Genetics 178: 1327-1337 [Abstract] [Full Text]  
• Choi-Rhee, E., Schulman, H., Cronan, J. E. (2004). Promiscuous protein biotinylation by Escherichia coli biotin protein ligase. Protein Sci. 13: 3043-3050 [Abstract] [Full Text]