Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 91, Issue 3, Pages 537-543Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2012.09.023
Keywords
Red (R)/ET (R) recombination; Homologous recombination; Chromosomal lacZ integration; Reporter plasmids; Promoter fusions; Gene expression
Categories
Funding
- Deutsche Forschungsgemeinschaft [Exc114/1]
Ask authors/readers for more resources
beta-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red (R)/ET (R) recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the X Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the gamma-origin-based suicide vector pNPTS138-R6KT, which can only replicate in lambda pir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K+-dependent activation of kdp promoter-lacZ fusions in vivo. (c) 2012 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available