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Antiobiotics for protein expression - (Mar/04/2011 )

Hi!

I would like to ask a simple question in order to determine if someone of you has such an experience. I am trying to express a Human enzyme in E.coli and the gene sequence of this enzyme contains many rare codons for e.coli. For that reason, I selected the strain BL 21 DE3 Rosetta gami 2. I got expression , but the yield is enough low (1.5 mg/L culture). It is stated that this strain has resistance for both chloramphenicol and kanamycin also. My vector (pET 14b) contains ampicillin gene resistance.
My question is simply: should I use both antibiotics for rosetta strain, e.g. ampicillin & chloramphenicol or just 1 is enough? will it affect the expression yield, or not? Do you have such an experience?

Thank you very much for your reply in advance.

All the best,

Chris

-Chris Max Planck-

When I use BL21 for expression and have Ap resistance on vector, I use Ap and Cm in overnight incubation. Then, when I inoculate medium for expression I use only Ap to insure, that cells do not loose the plasmid.

Nejc

-SOS response-

Hola, I would the prevention of use theese antibiotics if I prepare my own competents, but as Sos says, adding one of them in the preinoculum would be enougth to mantain episome (F), .I have use rosseta gami de3 pLys and only with the plasmid ab,(Ap) from the begining and the expression was ok.Its better work at 28 to avoid lost of episome. Buena suerte

-protolder-

thank you guys very much for your direct and informative replies.
If there is sth I could help you, just let me know.

-Chris Max Planck-