Nucleic Acids Research, Vol 26, Issue 16 3700-3706, Copyright © 1998 by Oxford University Press
FW Whipple
This report describes an Escherichia coli genetic system that permits
bacterial genetic methods to be applied to the study of essentially any
prokaryotic or eukaryotic site-specific DNA binding protein. It consists of
two parts. The first part is a set of tools that facilitate construction of
customized E.coli strains bearing single copy lacZYA reporters that are
repressed by a specific target protein. The second part is a pair of
regulatable protein expression vectors that permit in vivo production of
the target protein at levels appropriate for genetic experiments. When
expressed in a properly designed reporter strain, the target protein
represses the lac genes, resulting in an E.coli phenotype that can be
quantitatively measured or exploited in large scale genetic screens or
selections. As a result, large plasmid-based libraries of protein genes or
pools of mutagenized variants of a given gene may be examined in relatively
simple genetic experiments. The strain construction technique is also
useful for generating E.coli strains bearing reporters for other types of
genetic systems, including repression-based and activation-based systems in
which chimeric proteins are used to examine interactions between foreign
protein domains.
ARTICLES
Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. fwhipple@warren.med.harvard.edu
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