Dear Experts,
I was wondering if you could kindly provide some insight on how may I confirm a certain bacteria strain is BL21 strain whether biochemically or by molecular methods.
Your attention is highly appreciated.
Thanks,
You can possibly pcr for the missing genes or check for phage resistance to phage T1 by doing cfu counting... Other than that I am not sure how else you can check the strain.
https://www.neb.com/products/c2530-bl21-competent-e-coli#Product%20Information
Dear Kyle Skalenko,
Yes, the only significant marker that might help to validate this strain would be lack of two protease encoding genes.
Thanks for sharing your opinion.
yes, I agree. I would start there. If you can get your hands on phage T1 easily, then I would try a phage sensitivity assay by counting colonies and/or plaques.
Unfortunately, phage T1 is not available at our laboratory. I think PCR would be enough.
Are you talking about the parental BL21 strain, or the BL21(DE3) strain that expresses T7 RNAP? If the latter, then you can just PCR to confirm the presence of T7RNAP. Otherwise you can test for any specific genetic markers that are of concern to you.
Dear Michael J. Benedik ,
I highly appreciate your attention.
In my case, it would be better to confirm the BL21 parental strain. It seems lack of LON protease and OmpT encodings genes are the key markers for this strain.
If you want to confirm the protease mutations, those are all deletions so you can design primers to show the gene is missing (just be sure to amplify enough of the flanking regions to knwo that your PCR worked.
You might wish to check this article that I think does what you are wishing to do, and includes primer sequences:
Article A New Strain Collection for Improved Expression of Outer Mem...
Michael J. Benedik
I assume those missing proteases are the most prominent hallmarks of this strain, so they can serve as a validator for BL21.
Thank you so much for sharing the article!
if those protease deletions are in the strain then it’s most likely correct. If you really wish to confirm further the I would also verify T7 polymerase and that it’s a type B E. coli and not a K12. but it sounded like you mostly cared about the proteases anyway.
Michael J. Benedik
Indeed what matters, in this case, is to validate BL21(It has been used to produce a recombinant peptide). The approving agencies are asking for validation of the used strain. It seems there is no consensus method for this purpose so I must rely on genetics. Your suggestion for confirmation of B strain sounds logical as well. Thanks for the idea!
Validation for pharmaceutical production is entirely another matter. My recommendation is to send your production strain for whole genome sequencing, it shouldn't be that expensive and at least you know it will be sufficient for the regulators. Otherwise there may always be questions asked about how sure you are. And in the long run it will probably be faster for you.
Dear Michael J. Benedik
You are totally right, it is mentioned in the guidelines of FDA and other agencies that the production strain must be validated through WGS and supplier's documents. However, unfortunately, both are unavailable at the moment. If the regulators do not accept this method, there is no option but to perform WGS.