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Construction of stable, single-copy luciferase gene fusions inEscherichia coli

✍ Scribed by Angelina Guzzo; Michael S. DuBow

Publisher: Springer
Year: 1991
Tongue: English
Weight: 765 KB
Volume: 156
Category: Article
ISSN: 0302-8933
DOI: 10.1007/bf00245390

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✦ Synopsis

A ColE1-based plasmid for transcriptional gene fusions was constructed that contains both the promoterless luxAB genes of Vibrio harveyi and a tet marker within the inverted repeats of a left end-truncated Tn5 element. Introduction of this plasmid into an Escherichia coli strain containing a plasmid (pTF421) that over-produces ColE1 RNA1 (and thus inhibits replication of the ColE1 plasmid) allowed selection for cells that had a single copy of the luxAB operon transposed into the chromosome beginning 5 days post-transformation. The long latent period necessary for Tn5 transposition is analogous to that found in other systems, where transposition frequencies and mutation rates increase in a time-dependent manner when selected for upon prolonged incubation on Petri dishes under bacteriostatic conditions.

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