3 replies to this topic

[Chris Max Planck]

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

[SOS response]

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

[protolder]

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.It´s better work at 28º to avoid lost of episome. Buena suerte

[Chris Max Planck]

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