Repressed pTac leaks ! (Better ?)
jgraham at bronze.ucs.indiana.edu
Fri Mar 5 14:36:24 EST 1993
I have the pTac promoter upstream of a full lacZ in a transcriptional fusion
vector based on pSC101. The lac repressor is supplied for a single copy
lacIQ contruct carried as an F plasmid. The host is strain MV1190, a
recA derivative of JM101.
In the absence of any induction, B-gal levels of 800 Miller Units are
detected. With induction by 1mM IPTG, levels in the 6000+ Miller Units
are measured, with great variation between identical clones. Contructs
which include transcription terminators between the promoter and
reporter suggest that the transcription sufficient to saturate the B-gal
biosynthetic machinery are hit, leading to a breakdown in correspondence
between transcription levels and B-gal activity.
1) Have others driven a wild type lacZ copy from a very strong promoter
like pTac (Pr, ect.) ? If so, were B-gal levels proportional to
transcription under induced conditions ?
2) Is there a better promoter (met B ?) that can be repressed efficiently
and still give sufficient promoter activity for in vitro transcription
studies ? If so, where can I find it ?
Thanks very much,
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