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Construction of stable, single-copy luciferase gene fusions in Escherichia coli

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Abstract

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 overproduces 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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Abbreviations

Ap:

ampicillin

bp:

base pair(s)

kb:

kilobase(s)

PO:

promoter/operator

R :

resistant/resistance

Tc:

tetracycline

Tn:

transposon

xyl:

xylose

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Authors and Affiliations

  1. Department of Microbiology and Immunology, McGill University, 3775 University Street, H3A 2B4, Montreal, Quebec, Canada

    Angelina Guzzo & Michael S. DuBow

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  1. Angelina Guzzo
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  2. Michael S. DuBow
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Guzzo, A., DuBow, M.S. Construction of stable, single-copy luciferase gene fusions in Escherichia coli . Arch. Microbiol. 156, 444–448 (1991). https://doi.org/10.1007/BF00245390

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  • DOI: https://doi.org/10.1007/BF00245390

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