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Re: [ccp4bb] Bacterial induction at 18C |
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CCP4bb navigationCCP4bb <-- 2008 <-- April 2008 <-- 30 April 2008Subject: Re: Bacterial induction at 18C From: Gina Clayton gina {- dot -} clayton {- at -} YALE {- dot -} EDU Date: 2008-04-30 aside from the possibilites of toxic protein as already mentioned...... we had great results with overnight induction at 20oC for a protein that was somewhat insoluble at 37oC. One thing that might work for you is to grow the cells to OD 0.5 then lower the temperature to say 18 or 20. After an hour or so (retake OD) induce for overnight growth. Alternate is higher starting OD prior to temperature reduction (i.e greater mass of cells) and shorter growth time say 5-6 hours as you are thinking (we had protein that we only induced for 1 and a half hours too). Hope that is useful! Gina -------------- Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. CCP4bb navigationCCP4bb <-- 2008 <-- April 2008 <-- 30 April 2008 |
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