E. coli Rosetta (DE3) as well as E. coli BL21 (DE3) Codon Plus strains contain P15A-ori/CamR low copy plasmids, which provide extra copies of rare tRNA-genes. They are therefore used for expression of foreign genes with codons rarely found in E. coli. Although these strains are quite useful, they contain the extra tRNA-gene copies on plasmids. At the moment, I am assembling several genes to artificial operons in a combinatorial approach. I require the P15A-ori for one of my final constructs. Therefore, a strain analogous to the ones stated above but with the tRNA-genes integrated into the genome (rather than provided episomal) would be massively beneficial. Has anyone come across such a plasmid-free strain or are all of these strains equipped with ACYC-like plasmids?
I wouldn't use 'low copy number' to describe a p15A-based plasmid. pACYC and relatives often have copy numbers in the 15-25 range.
Anyway, my understanding is that the rare tRNAs genes contained in these plasmids remain under their native transcriptional control signals, and their increased levels in CodonPlus, Rosetta, etc. stem from their higher copy number when plasmid-borne. Wouldn't chromosomal integration eliminate this advantage?
Correct. In fact, these tRNA genes are actually present in the E. coli genome under control of the same promoters used for episomal expression. Therefore, as you state correctly, genome integration would not help. Given the number of E. coli strains generated for unique purposes over the last decades, it would be interesting to know if anyone has come across strains, in the genome of which tRNA-gene controlling promoters are altered to fit codon usage requirements.
I am not quite sure with the P15A-ori, but I used for my expression BL21(DE3)RIL. This strain I used for expression of plant genes in E. coli. This was my key strain for my diploma and thesis.
Johannes --- are you still in need of the pRARE2 with a different origin? If so, let me know at [email protected] .
Mark
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