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Archives of Microbiology

, Volume 156, Issue 6, pp 444–448 | Cite as

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

  • Angelina Guzzo
  • Michael S. DuBow
Original Papers
  • 52 Downloads

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.

Key words

Transposon5 Xylose operon Transcriptional fusions ColE1 replication luxAB genes Escherichia coli Vibrio harveyi 

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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Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Angelina Guzzo
    • 1
  • Michael S. DuBow
    • 1
  1. 1.Department of Microbiology and ImmunologyMcGill UniversityMontrealCanada

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