Stringent Lac promoter control

Sun Apr 24 13:32:00 EST 1994

	I have a cloned gene in plasmid pUC19.  I used an antibody
specific to the desired gene product to pick the clone from a
plasmid library.  The entire cloned fragment has been sequenced.
The clone includes a putative promoter, RBS, translation start,
coding sequence, and translation stop.
	I am getting low levels of expression from the cloned gene.
The gene is in the wrong orientation relative to the LAC promoter.
To increase production of the gene product, I attempted to force
clone the gene into the right orientation relative to the LAC
promoter.  This seems to kill the cells with or without the
presence of the IPTG in DH5alphas.
	DH5alphas do not produce enough lac repressor protein to
stringently control the Lac promoter on a  high copy number
plasmid such as pUC19.  As expected, test transformations with
pUC19 produced blue colonies on selective medium with or without
	I switched from DH5alphas to JM109s.  JM109s are a mutant
strain of E. coli that over produce the lac repressor protein.
I was surprised when test transformations indicated that I still
did not have stringent control over the Lac promoter.  Test
transformations yielded blue colonies with or without IPTG.
I then "ass u me d" that JM109 cells must only be effective for
providing strict control of the lac promoter in low copy 
expression vectors (???).
	I purchased a high copy number plasmid similar to pUC19
(alpha complementation), but which also expresses the lacIq
gene from each copy of the plasmid itself.  Test transformations
in DH5alphas indicate that I still do not have control over the
lac promoter.  Now, I get WHITE colonies with or without IPTG.
I have tried doubling the concentration of IPTG.  Ampicillin
resistance indicate that the cells have plasmids.  Blue colonies
from pUC19 transformed DH5alphas indicate that the Xgal is OK.
	Please critique my analysis and/or provide suggestions.
Thanks for your time and kind help in advance.
Charles Krueger  Krueger at

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