need help on acid plate screening
tw26 at cornell.edu
Tue Sep 19 09:20:00 EST 1995
Dear netters. Thanks for those who responded to my previous message. I
have another problem though.
I am trying to clone a gene coding for UDPG:fatty acid glycosyltransferase
from a wild tomato species. The enzyme transfers glycosyl group from UDPG
to fatty acid to form acylglucose. Since we experinced an extremely
difficut to purify the enzyme to homogeneity we decided to clone the gene
by directly screening cDNA expression library using pYES2 as a vector.
The screening strategy we designed is based on observations by Stevens
(1993) and Viegas (1989), that it, the inhibition of yeast growth by the
acids is believed to result from depletion of H+ gradient across plasma
membrane by an acid-induced higher rate of H+ influx. Assuming that
glycosylation can detoxify the fatty acids, we designed the following
screening protocol: yeast transformants with cDNA library are selected on
SC-ura plates and then replicated onto YP-galactose plates plus 10 mg/L
decanoic acid. We have tried this many times and haven't got any positive
clones. I am wondering if our strategy is physiologically or molecular
biologically unreasonable. Any comments and advices will be greatly
appreciated. Thanks in advance.
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