expression plasmids for tRNA
Posted 17 December 2012 - 02:07 AM
Has anyone done tRNA overexpression? I came across very old papers in this regard, I don't know if some plasmid vectors are available for tRNA expression. I am just reading literature and going through the papers, completely new to RNA work. Please help me experts out there. Need to overexpress tRNA in E.coli for further studies.
Posted 18 December 2012 - 06:12 PM
Posted 18 December 2012 - 08:38 PM
I think, this template is for his-tRNA, i don't know if I am right ..can it be used for other specific tRNAs....also look at the paper-
this is quite old paper....I dint find any recent paper related to these kind of studies.
Posted 19 December 2012 - 02:41 AM
The sequence for those plasmids may be online, although I'm not certain it is. Even if not, it would be easy to sequence it. There is nothing difficult about cloning a tRNA gene -- it's just like any other gene, except the end is determined with a transcriptional terminator. The mRNA is processed after transcription by precise cutting, so the flanking mRNA bases must be present.
The first reference was for production of tRNA in an in vitro system, and is probably not what you want. The second is much closer, and should show you how to construct the desired plasmid. You may or may not need the overexpression at high temperatures (I probably would avoid that complexity).
Posted 03 January 2013 - 12:35 PM
Our lab has cloned tRNA into pUC18 vectors with success. For in-vitro transcription, we insert the vector into DH5-alpha and then mega prep the plasmid. We then cut with BstNI (cuts after CCW), in vitro transcribe, run on a Urea gel, then extract the tRNA from the gel and do a final ethanol precipitation to clean it all up. I have done some purification of native tRNA but for my purposes it isn't totally necessary. I am not as familiar with the protocol to do that so I'm afraid any advice I may give you might turn out to be bad advice. I'll have to ask my lab mates more about the native tRNA purification protocol.
Recently, a labmate of mine spoke with another lab that uses a vector that will self-splice the inserted gene, and I believe they can purify both native and in-vitro transcribed tRNA without having to do any restriction digests, which is nice. Again, I don't know much about this vector (the name of which escapes me), but if you are starting from scratch, it may be easier to start this way.
Posted 03 January 2013 - 08:03 PM