B.subtilis beta-gals

Joe Sarsero jsarsero at cmgm.stanford.edu
Thu Nov 23 04:18:23 EST 1995


     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  *
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