Repressed pTac leaks ! (Better ?)

the End 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.

Two questions:

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,

Jim



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