Q: Chloramphenicol Resistance

Scott Narimatsu narimats at usc.edu
Fri Jun 20 14:49:38 EST 1997


We have been working with the pbunicat gene and the bssk vector in
attempt to insert the CAT gene into the bssk vector.  We completed a
blunt end insert with cuts at Nco I and Nsi I in CAT and Sma I in bssk. 
The CAT insert remained in frame with the b-gal start site and still
retained its stop codon.  We used blue/white selection to find
transformed cells and restriction digest to find ligated plasmids with
correct orientation.  We then tested for CAT resistance by first using a
one hour incubation time for IPTG and differing concentrations of
chloramphenicol from 12.5 ug/ml to 100 ug/ml.  No visible growth
resulted after over 24 hours of incubation at 37 C.  We then set the
chloramphenicol concentration at 12.5 ug/ml and altered the IPTG
inoculation time from 1 to 4 hours.  After over 48 hours of incubation
at 37 C, there was no visible growth.  However, after leaving the
cultures at 4 C for 24 hours, significant growth occurred.  Analysis of
the concentration of bacterial cells showed a direct positive linear
relationship between incubation time with IPTG and concentration of
cells.  If anyone has any idea or information on these type of results,
please write back.  We have considered the fact that the leader strand
before CAT from b-gal, may be inhibitory.

Thanks,
Scott



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