Thanks for your response. I am expressing PFK in
bacteria that lack PFK. Thus, my protein should
not only NOT be toxic, it should rescue the
bacteria so they can grow on glucose. I also
have this problem with an empty plasmid only
coding for EGFP. I am doubtful this is toxicity.
I am getting conflicting stories about requirement
for SD sequence. On the one hand, Novagen and
others claim it is obligatory. On the other hand, a
lot of people do not have this in their inserts,
claiming it is unnecessary. However, checking a
couple plasmids, we discovered the presence of the
SD sequence within the plasmids they used (specifically,
AGGAGG). Therefore, SD is not required in many
inserts, but only because it is present within
the plasmid. Our failed attempts have been
using both plasmids and inserts both lacking SD.
Could you please confirm that you get high protein
expression using a plasmid AND insert which both
lack the AGGAGG sequence and uses T7 promoter?
Thank you very much for your time. It is