I have recently begun to work with Bacillus subtilis for the first
time. I am looking at gene regulation and often utilize lacZ fusions to
study gene expression. I am working with some temperature-sensitive
alleles and need to grow the strains at 30C and 42C. A problem that I have
encountered is that beta-galactosidase activity at the higher temperature
is approximately ten-fold lower than at the low temperature. This occurs
in otherwise wild-type strains (no ts allele present), and has happened
with three totally different promoter-lacZ fusions. It therefore appears
to have something to do with the beta-gal enzyme itself rather than
properties of the fused promoters.
All of the fusions I am using are translational fusions. Strains are
grown to OD600 of 0.4 to 0.6 in Vogel-Bonner minimal medium, and beta-gal
activity is assayed at 28C according to the standard Miller protocol.
Is this a common occurrence in B. subtilis or am I doing something
drastically wrong? Thanks for any suggestions.
Joe
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* * *
* Joseph P. Sarsero, * *
* Department of Biological Sciences, * Ph: 1 415 723-1141 *
* Stanford University, * 1 415 723-3165 *
* Stanford, California, 94305-5020, * *
* U.S.A. * Fax: 1 415 725-8221 *
* * *
* *
* E-mail: jsarsero at cmgm.stanford.edu *
* jsarsero at leland.stanford.edu *
* WWW: http://www-leland.stanford.edu/~jsarsero/ *
* *
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