An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures.
Placing a gene of interest under the control of an inducible promoter greatly aids the purification, localization and functional analysis of proteins but usually requires the sub-cloning of the gene of interest into an appropriate expression vector. Here, we describe an alternative approach employing in vitro transposition of Tn Omega P(BAD) to place the highly regulable, arabinose inducible P(BAD) promoter upstream of the gene to be expressed. The method is rapid, simple and facilitates the optimization of expression by producing constructs with variable distances between the P(BAD) promoter and the gene. To illustrate the use of this approach, we describe the construction of a strain of Escherichia coli in which growth at low temperatures on solid media is dependent on threshold levels of arabinose. Other uses of the transposable promoter are also discussed.
Grant
A J
AJ
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton S016 7PX, UK.
Haigh
R
R
Williams
P
P
O'Connor
C D
CD
D001089:Arabinose / Q000096:biosynthesis / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002455:Cell Division / Q000187:drug effects; D004251:DNA Transposable Elements / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D009876:Operon / Q000235:genetics; D010750:Phosphoproteins; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D013696:Temperature
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D010750:Phosphoproteins; D001089:Arabinose; D020558:GTP Phosphohydrolases; C408370:typA protein, E coli