I recently received a Rosetta strain from a university research group, along with some expression constructs. When I asked for chloramphenicol to maintain the pRARE plasmid carrying the rare tRNA genes, I was told that they routinely cultivate the strain without any chloramphenicol. Surprised, I asked whether that would lead to plasmid loss over time, but they responded that, in their version, the rare tRNA genes are integrated into the genome.
However, I have not been able to find any documentation or commercial source supporting the existence of a Rosetta strain with genomically integrated rare tRNA genes. As far as I am aware, all known Rosetta derivatives rely on plasmid-based expression systems (e.g., pRARE, pRARE2), which require chloramphenicol selection for stability.
Could anyone clarify whether such a genomically modified Rosetta strain exists, or if this might be a misunderstanding?
They are risking ccDB mutation "complications" by not including the chloramphenicol.
You can have the genome sequenced to see if the rate tRNAs are in the genomic DNA. I'd sequence the plasmids too.
Never trust plasmids (or cells) that you get from someone else. Sequence all of it. Mistakes are easy to make (all tubes of bacteria look the same, labels get smeared, etc.).
Good luck!
And please write back when you figure out what's going on with the location of the rare tRNA genes
it is possible that in fact their expression constructs do not need the rare tRNAs and even if they have lost them (by not applying chloramphenicol resistance) they don't see it. Is the bacteria they provided chloramphenicol resistant? as Katie wrote "Never trust plasmids (or cells) that you get from someone else" buy some rosetta cells from the supplier (could save lot of time even if they are expensive...)
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