1887

Abstract

We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in simply and rapidly. This system takes advantage of an shuttle vector that contains the replication region of the theta-mode multiresistance plasmid pSK41, and is therefore a stable low-copy-number plasmid in the latter organism. This vector also contains a multiple cloning site downstream of the IPTG-inducible P promoter for insertion of the gene of interest. Production of encoded proteins can be stringently regulated in an IPTG-dependent manner by introducing a pE194-based plasmid, pGL485, carrying a constitutively expressed gene. Using GFP fusions to two essential proteins of , FtsZ and NusA, we showed that our plasmid allowed tightly controlled gene expression and accurate localization of fusion proteins with no detrimental effect on cells at low inducer concentrations. At higher IPTG concentrations, we obtained sixfold overproduction of protein compared with wild-type levels, with FtsZ–GFP-expressing cells showing lysis and delocalized fluorescence, while NusA–GFP showed only delocalized fluorescence. These results show that our system is capable of titratable induction of gene expression for localization or overexpression studies.

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2011-03-01
2019-08-26
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