Hello! Are you sure that you need that strain of E. coli? If you know the sequence of your transgene you can use the codon database to see if it will be expressed without issue. Rosetta allows for an expanded range of translation. Try the 'graphical codon usage analyzer" here, https://gcua.schoedl.de/ You may need the PLysS, but you can find DE3 E. coli with PLysS fairly easily. If the codon usage is bad, you may have to swap it out with quickchange mutagenesis. Also, unfortunately when you change the construct and strain you most likely will have to do expression testing over again. Good luck!

have a look for BL-21 and versions thereof (Rosetta is basically that anyway). I have used the 'codon plus' variety which has an older pRARE plasmid to encode rare tRNAs in E.coli (its only one fewer tRNA than found in Rosetta strains). Much of the time you dont even need the rare tRNAs, and nowadays some proteins are thought to be better off being expressed slowly on the ribosome rather than as fast as possible.

Thanks Matthew Edward Thornton for your response an suggestion. Our collaborators actually used that strain for Cas13a expression and we just wanted to reproduce the result using the same strain. I have DE3 so I will try to express the protein in it and will also check the codon usage using the link that you have provided. Thanks once again :)

Gary James Hunter Thanks for your suggestion. I have BL21 (DE3) so I will try expressing the protein in it. There is another alternative called Rosetta 2(DE3)pLacI. I am thinking of trying that too.

Hello Subha, I would highly recommend the SixPack strain from the lab of Dr. Zsuzsanna Gyorfy. Genes encoding rare tRNAs are encoded in the genome, no chloramphenicol needed to maintain an additional plasmid. See publication below. Good luck!

Article Enhancing the Translational Capacity of E. coli by Resolving...

Hi Jamison Drew Law Thanks a lot for your response and suggestion. I will read the paper and try to get the vector from Dr. Gyorfy's lab.

Kind regards,

Subha

Subha, to be clear SixPack is a strain of E. coli, not a plasmid. Good luck!

Hi Jamison Drew Law Sorry for the typo. Yes, indeed. The strain :)