Hello,
my electrocompetent E.coli BL21 (DE3) cells are having low transformation efficiency with pAC28 vector-insert (10 - 30 kb). It was around 1 × 10^4 and less / 1microgram of DNA.
Pladmid-insert was isolated from DH5a cells by QIAQEN MAXI kit. I prepare cells for electroporation according to attached protocols.
I would be very thankful for your experience! ;)
https://www.lucigen.com/docs/manuals/MA145-ClearColi-BL21-DE3-Electocompetent-Cells.pdf
For such an enormous vector, you can't expect much more from BL21. This strain is optimized for protein production, not for transformation efficiency. Usually people need only one colony and they're good.
What's more typically done is that you prepare libraries in a DNA propagation strain, in the case of big inserts, 10beta would be best. And then you select some kind of "winner" for expression in BL21.
Is there any particular reason you need BL21 for this? Even with normal sized inserts I see its transformation efficiency being some 100 fold lower than that of the DNA strains.
I agree with John. I would suggest you to go with E.Coli. DH5α starin using ultra comp. cell preparation protocol provided by CSH.
CSH Protoc. 2006 Jun 1;2006(1). pii: pdb.prot3944. doi: 10.1101/pdb.prot3944
Best of luck...
I agree with John and Eshan, BL21 strains do not have high transformation efficiencies but normally it is OK if you get a few clones. I would also prefer a different protocol than electrocompetent cells, they are sometimes difficult to prepare and to handle.
What origin of replication does pAC28 have? You should also check whether it is compatible with your Rosetta plasmid. Furthermore, the plasmid is pretty big and I guess you want to express a protein so you use BL21 strains. You should keep in mind that E. coli is not very efficient for the expression of very big proteins.
If you need an expression strain with high transformation efficiency as well, you can use E. coli KRX (Promega) which are K12 strain derivatives with T7 polymerase in the genome under control of arabinose promoter, so you can use autoinduction for expression. Then, just isolate your Rosetta plasmid from your BL21 strains, put this in KRX first and prepare you competent cells. Haven't tried something like this yet, but I guess it could work.
Hello, thank you for sharing your knowledge :) They helped me a lot! I would like to respond to some of your questions:
John, thanks for advice. I chose it for making a library that I can screen by functional screening as long as it is good type of host for heterologous expresion for expresion of certain proteins. But I am aware of their low transformation efficiency. I hoped there would be some protocol for enhancing their efficiency.
Eshan, thanks for advices and protocol info., I will see how it is working with our cells ;)
Jonas, thanks for your advices. pAC28 has p15A ORI. It is compatible with BL21 (DE3). I would like to express protein of cellulase from metagenomic DNA at least into cytoplasm of Rosetta. It should be possible for this kind of protein according to few articles (PMID: 26452465; PMCID: PMC4600292). Do you have different experience?
According to the title of the question, you use E. coli BL21(DE3)Rosetta cells and not only regular BL21(DE3). The Rosetta genes for rare codons are encoded on the pLysS plasmid which has the same origin as pAC28 (p15A). Thus, your pAC28 is not compatible with the Rosetta plasmid as they share the same ori. Do you select for the Rosetta plasmid with chloramphenicol after transformation? Maybe the compatibility classes of your plasmids are a reason for your low transformation efficiency. If you don't need the Rosetta plasmid for the expression of your construct of interest, just use regular BL21(DE3) cells, they probably grow better.
If there are several protocols published it probably works but your vector is pretty big as John stated already that's why I assumed the protein might be too large to express in E. coli.
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