The purpose is cell based functional assay for the recombinant protein not protein purification.
No, LMG194 does not express the T7 polymerase so a pET vector will not drive transcription to any significant level. However there is likely to be some low level expression of the cloned gene, anytime you have a gene on a multicopy plasmid you will have sufficient random transcription to see some (but low) expression.
DEar Nitesh Kumar Khandelwal
as Michael J. Benedik told you since the LMG194 miss the T7 polimerases, it cannot express protens in this strain.
But there are other vectors based on lac or TAC promoters that may work in this strain.
best regards
Manuele
I should add two things, first you can make a derivative of LMB194 with the T7 polymerase but this requires some knowledge of E. coli genetic manipulations. Secondly, LMB194 is not really a particularly special strain, is there a reason you wish to particularly use it instead of some other strain?
Michael J. Benedik
Hi Michael,
Thank you for the reply.
I want to use one derivative of LMB194 strain in which multiple membrane transporters are deleted.
My protein of interest I expressed in BL21 with pET vector and I have multiple mutant variant of this protein in pET vector. I wanted to perform some transport assay with LMB194 derivative strain using my protein mutants variants.
If pET vector could express any leaky level in LMB194 transporter knockout derivative strain. It could have been very easy to perform transport assay without switching vector for all the mutant variants of my protein.
Best
Nitesh
It might work for what you need, you will just need to try.
If not, you can probably clone into any blue/white screening plasmid with a lac promoter, should be easy enough to clone your mutants unless you have lots
Michael J. Benedik
Hi Michael,
I am going to try with my pET vector and will confirm by western blot if I could see some leaky expression.
Thank you for your suggestion.
Best
Nitesh
Manuele Martinelli ,
Thank you Manuele for the response.
Best
Nitesh
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