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OD and growth rate as indicator of protein expression?


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#1 miche

miche

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Posted 21 February 2011 - 04:46 AM

Opps, sorry, this topic was already started (here and here, but I posted too quickly. I'm happy to merge this text with the relevant other posts if necessary.
Thanks
M

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Hi there,

I made few constructs to express a gene of interest (a 11KDa protein) in E. coli.
I use chemically defined medium and 1mM IPTG induction.

As empirical rule to check expression, I measure OD and growth rate for the induced/uninduced samples: after successful induction growth rate drops and OD remains low.

Does anybody could confirm that if OD and growth rate for the induced/uninduced conditions do not change, means no expression (hence no waste of gel)?

I was able to express my construct only with pET and BL21(DE3). I want to move away from T7, but so far I never had luck (i.e.: no change in OD and growth rate), including a commercial vector (hence with well formed structure). My ideas for trouble shooting are make new IPTG stocks, and transform my plasmids in the good old DH5a and MG1655.
Is T7 so effective that other system do not work at all?

Does anybody have some more ideas on troubleshooting?

I'll keep you posted as soon as I run few more experiments.

Many thanks!
Miche

Edited by miche, 21 February 2011 - 05:27 AM.





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