narimats at scf.usc.edu
Thu Jun 19 18:31:37 EST 1997
I have been transforming the CAT gene under the lac promoter into
sensitive bacteria and selecting for chloramphenicol resistance. We
ligated the CAT gene into the multiple cloning site of pBssk. The CAT
gene remained in frame with b-gal and the CAT gene still had its stop
codon. We transformed the plasmids and used blue/white selection to
select for the CAT insert and used restriction digests to select for the
correct orientation. We then selected for positive chloramphenicol
reistence. our first test used a one hour incubation time with IPTG and
then differing concentration of Chloramphenicol from 12.5 to 100 ug/ml.
Overnight incubation at 37C showed no observable growth. We then tested
with differing IPTG incubation times from 1-4 hours at a chloramphenicol
concentration of 12.5 ug/ml. After 48 hours at 37C shaking, there was
again no observable growth, however, following the 48 hours, the samples
were placed at 4C. Upon checking the bacterial samples after 24 hours,
they showed significant growth in all tubes. After analysis by
spectrophotometry, there was a definite positive linear relationship
between the incubation time and growth rate. Ideas that have arisen are
the inhibition of the CAT protein by the leader strand of b-gal, but
this does not explain many of the other phenomenon. Any suggestions?
Thank you for your time.
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