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problem with a galactose based screen

MICHELLE BALLARD ballarmj at lldmpc.dnet.dupont.com
Fri Oct 13 14:11:24 EST 1995


I am working on a project that is designed to identify fragments
of a gene which when coexpressed with the full length gene will
inhibit its function in a dominant fashion.  I have the full length
gene constitutively expressed on a CEN plasmid (GPD promoter) such
that it complements a temperature sensitive mutation.  The fragments
are on a multicopy plasmid (2um)with regulated expression via the
GAL 1-10 promoter.  I identified by relpica plating several colonies
which grew on glucose but not on galactose. I isolated the 2um plasmid
with the fragment and retransformed the original strain to see if
the no growth phenotype would be maintained.  The newly transformed
strain will grow on galactose but forms very tiny colonies as compared
to those on glucose or the control colonies (transformed with 2um plasmid
without insert) on galactose.  I can't figure this out.  There is still
growth inhibition, but it isn't as it was in the original strain where
there was absolutely no growth on galactose. Could there be a copy number
problem? Any ideas, thoughts, or suggestions would be greatly
appreciated. I'm sorry to avoid the name of the gene in question, but
it's considered proprietary (I can talk about it, but I can't broadcast
it over the net - company rules).

			Thanks in advance!!

			Michelle Ballard

e-mail:  ballarmj at lldmpc.dnet.dupont.com



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