Construction of stable, single-copy luciferase gene fusions in Escherichia coli.
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.
Guzzo
A
A
McGill University, Department of Microbiology and Immunology, Montreal, Quebec, Canada.
DuBow
M S
MS
D003001:Cloning, Molecular; D004261:DNA Replication; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005822:Genetic Vectors; D008156:Luciferases / Q000235:genetics*; D009876:Operon; D010957:Plasmids; D012329:RNA, Bacterial / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D008156:Luciferases