I am thinking of buying a ColE1-compatible plasmid to co-express biotin ligase (BirA) in E. coli, with the intention of biotinylating in vivo our Avi-tagged constructs. Whatever we end up buying (or constructing) must be compatible with the use of autoinducing media as well.
The problem is that so far, in every commercially available plasmid I have found, BirA expression is driven from a lac-derived or AraB-derived promoter, and our expression constructs use a T7 promoter. So, in autoinducing media BirA transcription would start simultaneously with that of the T7 RNA pol gene, meaning that the BirA transcripts would have to compete for translation with the onslaught of T7-driven transcripts. I suspect that this would lead to less than ideal levels of biotinylated product, and the problems reported for instance in PMID 22227598 seem to confirm my suspicion.
So, my question is, a) is this really a problem?, and if it is, b) Can it be solved by using a different promoter for BirA coexpression, or should I simply do biotinylation in vitro with purified BirA?
Dear Alejandro,
I assume that the problem you refer to in the paper mentioned relates to accumulation of BirA in inclusion bodies during expression? I agree that this could limit the amount of the biotinylated target protein produced, but i dont think the actual outcome can be predicted easily. The authors also state that 1:100 BirA:target ratio was enough to fully biotinylate the target protein in vitro, which could suggest that even if you're birA expression is severely throttled it may still be enough to ensure your target protein is biotinylated. I would suggest to just try it out. If it doesn't work you can still use your BirA plasmid for expression, or as a template to clone a tagged BirA as per the paper you mention.
Hope that is useful.
Silas
Hi Silas, thanks for the help!
Actually, I was referring to the fact (stated on the introduction of the paper) that they get low and variable levels of in vivo biotinylation when they use Avidity's pBirAcm together with T7-based plasmids.
- Badges
- Science topic