Dear D. 

As far as I know, E. coli strains derived from K-12 do not carry such a feature. Instead, you should try with E. coli BL21 DE3 which, in turn, is derived from B strain and is commonly used to express T7 promoter-driven genes. 

Good luck.

Thank you, Angel.

I'll check this.

Daniel,

I concur with Angel. In our lab we have used T7 promoter plasmids (pET vectors) with great success within the BL21 (DE3) cells. High rate of protein expression.

Daniel,

You 'd better try host strains which carry DE3 gene a.k.a. T7 RNA polymerase, like BL21(DE3) or Rosetta(DE3)

Thank you, Guys.

The problem is that I want to complement this sod mutant with a gene from another bacteria.

It's not to recover the protein, it's just to see if this other is complementing this specific K-12 mutant. That's why I'd like to use it.

Daniel,

From my experience, I used a Keio Strain e.coli (a K-12 strain with a knocked out gene of interest). Since K-12 strain is not suitable for expression, I had to lysogenize it with a bacteriophage. In this case, my gene is in a pET-15b plasmid (with a T7 promoter) so I had to use a DE3 T7 Phage to infect my keio strain for it to have an RNA polymerase gene. It worked for my mutant enzymes expression. 

There's another study that used this Keio Strain (K-12) in expressing several variants of TIM (Triosephosphate isomerase).

https://chemistry.osu.edu/~magliery/pdfs/SullivanMagliery2011JMB.pdf

I hope this helps.

Thank you, Archie. I'll check this!!

Regards.

Hello Daniel,

having a similiar problem I found this thread via google search. I also try to introduce a plasmid with T7 promotors (pETM6) in an already genetically modified E.coli K12 strain. Hence, I am interested if and how it worked for you.

I am also glad for input from other people.

Best regards,

Raphael

Dear all,

it is quite challenging, but possible to integrate the T7 polymerase gene into the chromosome of your K12 strain of interest, and thus use the strain for T7 based expression. For more information please contact me directly via: [email protected].

Bests, Marlen

You can deliver the T7 RNA pol gene on an engineered bacteriophage vector. Grow the cells, then infect them at a moi high enough to guarantee 100% infection and induce; no need to prepare DE3 lysogens or use special strains. Novagen (now Merck Millipore IIRC) sells a lambda phage called CE6 for exactly this purpose; look it up to see if it suits your purpose.

Ok, I know this is 2-3 years old, but pulls out from google searches

@ Marlen Schmidt

"and thus use the strain for T7 based expression. " Sorry, from my own experience and others, most K12 strains do not perform well as DE3 derivatives. T7 RNA polymerase is 5x faster than E. coli polymerase (the ribosomes have the same speed as E. coli polymerase); hence "naked" mRNA are produced by T7 RNA polymerase and - with the much higher nuclease content of a K12 strain - most mRNA is degraded before the protein is expressed. Church lab @Harvard uses T7 RNA polymerase expressed from a plasmid (higher T7 RNA polymerase levels); this partially works, but stressed the K12 strain much. We got it work on the mentioned Keio BW25113 strain; deletion of carboxyterminal domain of RNase E helped.

@Daniel Chaves de Lima Please keep it simple; I have had this problem several times; the easiest solution for me was to exchange the T7 promoter by the tac promoter via inverse PCR . Amplify the complete plasmid as linear PCR product introducing the changes through the 5´-ends of the primers. Gelpurify the PCR product, use 100 ng for T4 PNK and T4 DNA ligase in DNA ligase buffer, heat inactivate, DpnI digest and transform.

I am in same boat @Markus Englert, does tac promoter works well with your knockout K12 expression strain? I am trying to express a protein in knockout mutant host, the original vector has T7 promoter, so just want to minimize my efforts figuring out which promoter would work in my new host.

Thanks

Sanjay

What about the T7 terminator then? Can it work same as tac terminator? @Sanjay Patel @Markus Englert

Hello, late answer/ question. I am about to clone my gene from pET vector (T7 promoter) into pBAD (arabinose promoter) to avoid this problem and then electroporate into the KEIO strain. I THINK the strain has a deletion in the arabinose pathway, so pBAD arabinose-inducible promoter should be useful to express this protein of interest in the KEIO strain. The expression might be lower than with T7 RNAP but the whole process will be easier than making a chromosomal mutation in the strain.

Feedback appreciated,

Christina