Repressed pTac leaks ! (Better ?)

G. SCOTT GORDON ggordon at opal.tufts.edu
Mon Mar 8 02:02:00 EST 1993


In article <C3FL4p.6yu at usenet.ucs.indiana.edu>, jgraham at bronze.ucs.indiana.edu (the End) writes...
>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
We've actually run in to a similar stumbling block in our lab 
(eg. even lac i-q allows a significant basal level of transcription
from P(lac) or P(tac)).  In our case we also wish to assay for
B-galactosidase "ON" or "OFF" on X-gal plates. Using low copy
number (approx. 10 copies/cell) lac Z with normal P(tac) we still
get significant background blueness.  
		You may be interested (as we were) in a Boerenger-Mannheim
vector
called pEX (1,2, and 3) ($87).  It's basicly a full lac Z gene under the

control of the lambda right arm promoter (Pr).  It was designed 
to make B-gal fusion proteins (MCS after lacZ C-term). 
		While I don't know the Miller Unit levels for this Pr-lacZ
fusion,
I DO know that a similar fusion with a crippled repressor binding site
(Or-2 specificly destroyed) gives Miller units on the range of 100
(reference: Amster-Choder and Wright, Science: 257: p.1396 Table 1).
I can only assume that wild-type Pr would allow even better 
repression.  Also, my PI tells me that 100 miller units is the border
between SEEING and NOT seeing blue on Xgal plates.  Further, Pr is
considered quite a strong promoter when not repressed.
		Anyways, hope this information helps you (as well as me).

														
Good luck!

																			
Scott Gordon
																			
Tufts University
																			
Sackler School of Biomedical Sciences				



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