full length lacZ ?

the End jgraham at bronze.ucs.indiana.edu
Thu Dec 9 18:12:09 EST 1993


Russ,

When you get that plasmid, you will have very careful as full length
lacZ on a plasmid is a very unstable situation. Although this must
be widely known, it is not widely reported. In a course we offer here 
students merely grow an E.coli strain containing pSKS105 and collect 
lac deletion mutants that arise at a high spontaneous frequency. 

Athough a single copy lacZ on a phage or intergated into the 
chromosome might be an alternative, I would seriously consider an
alternative reporter gene (eg. galactokinase, luciferase, cat, ect)
which will unfortunately be more difficult to assay. Another problem you
may see is that even with a pUC copy number of lac repressor, the
basal level of B-gal activity remains high enough to create blue 
colonies on X-gal plates. An alternative like the weaker arabinose
promoter can reduce this uninduced level, though still not below
the X-gal threshold.

pSKS105 is from:

Shapira, SK, Chou, J., Richaud, F.V., and M. J. Casadaban. 1983.
Gene 25, 71-82.


Jim
J. Graham
Indiana Unviersity



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