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

摘要

Cosmids are plasmids that contain the phage λ sequences (cos) required for packaging of the phage DNA into the virion. Induction of a λ 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.