An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures
Grant, A.J., Haigh, R., Williams, P. and O'Connor, C.D. (2001) An in vitro transposon system for highly regulated gene expression: construction of Escherichia coli strains with arabinose-dependent growth at low temperatures. Gene, 280, (1-2), 145-151. (doi:10.1016/S0378-1119(01)00769-7).
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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 TnΩPBAD to place the highly regulable, arabinose inducible PBAD 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 PBAD 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.
|Keywords:||promoter, Tn5, transposition, expression system, BipA|
|Subjects:||Q Science > Q Science (General)
R Medicine > R Medicine (General)
|Divisions:||University Structure - Pre August 2011 > School of Biological Sciences
|Date Deposited:||08 Aug 2008|
|Last Modified:||12 Jul 2013 01:05|
|RDF:||RDF+N-Triples, RDF+N3, RDF+XML, Browse.|
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