Hi, Can you explain what you want to do?
Do you just want more of some tRNA corresponding to a codon that is rare in E. coli you can use the pRare2 plasmid that you can easily isolate from the Rosetta2 strain (or use Rosetta2 directly if you don't mind). If you just want some extra tRNA production for the AUA, AGA and AGG codons, there is also the pSJS1240 plasmid from the Kim lab that you can get from Addgene: http://www.addgene.org/pgvec1?f=c&cmd=findpl&identifier=12234 You are right that an induced T7 promoter will give way too much tRNA. It will not only be wasteful, the tRNA modification machinery – that is needed to make the tRNA fully functional – will not be able to keep up and this may create havoc. Cheers, Martin On Nov 16, 2010, at 12:00 AM, Patrick Loll wrote: > Here's an ignorant question: When people express an exogenous tRNA in E coli > (to overcome rare codon issues, for example, or to supply a cognate tRNA for > an orthogonal synthetase), what sorts of promoters are used? > > My (ignorant) guess is that something as potent as the T7 promoter might be a > waste, since you don't need huge quantities of your tRNA, and you probably > don't want to divert resources away from message production for your target > gene. But I could be wrong. > > Any pointers to sequences that successfully direct tRNA production would be > welcome, so I could use them as design templates. > > Thanks, > > Pat > --------------------------------------------------------------------------------------- > Patrick J. Loll, Ph. D. > Professor of Biochemistry & Molecular Biology > Director, Biochemistry Graduate Program > Drexel University College of Medicine > Room 10-102 New College Building > 245 N. 15th St., Mailstop 497 > Philadelphia, PA 19102-1192 USA > > (215) 762-7706 > pat.l...@drexelmed.edu
