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[Arabidopsis] why plant TF gene clone failed to grow in DH5alpha?

Smith Chiang via arab-gen%40net.bio.net (by chiangscn from gmail.com)
Mon Jul 20 20:35:08 EST 2015


hello colleagues,

Recently and two years ago, we met a wierd problem when growing clone
containing plant transcription factor (TF)gene  in the commonly used
E.coli strain DH5alpha. That is, after we cloned the the coding region
(CDS) of TF genes into a yeast two-hybrid vector and screened out
positive clones, we want to extract plasmid from overnight cell
culture after innoculating single colony into LB(+50 mg/ml Kan).
However, among several cell cutures harboring different TF
genes(belong to the same family), one did not grow at all (medium
transparent after O/N shaking),while all others grow well. This
occured two years ago when we worked another TF gene in Y2H vector
(shuttle vector, propogate in both E.coli and yeast). However, colony
PCR indicates that it is the right clone.

I think Y2H vector plasmid only express antibitic resistant gene, and
propagate in E.coli through DNA replication, no transcription or
translation of target gene(i.e. GAL4-BD-TF fusion gene) in E.coli,
right? So, it is not because expression of TF that caused non-growth
of E.coli, right? Or is there leaky expression of GAL4-BD-TF under ADH
promoter in E.coli? BTW, we used Clonetech Matchmaker Y2H system in
our lab.

Am I missing anything? Or does this tell us something about the
function of the two TF genes?

thanks for your reply.

Best regards,
Smith



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