Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

expression plasmids for tRNA


  • Please log in to reply
5 replies to this topic

#1 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 17 December 2012 - 02:07 AM

Hi,

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.


Thanks

#2 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 18 December 2012 - 06:12 PM

Several protein expression system have plasmids overexpressing tRNAs, especially rare codon tRNAs. Check the Origami and Rosetta strains of E. coli BL21 (DE3).

#3 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 18 December 2012 - 08:38 PM

Thanks Phage, you are right that these strains are having rare codon expressing tRNAs, but I was wondering if tRNA can also be cloned like any other gene, and if it can be expressed in some plasmid, for instance have a look on this link-
http://www.kerafast....-in-e-coli.aspx

I think, this template is for his-tRNA, i don't know if I am rightPosted Image ..can it be used for other specific tRNAs....also look at the paper-
http://www.ncbi.nlm....i tRNA”“’

this is quite old paper....I dint find any recent paper related to these kind of studies.

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 19 December 2012 - 02:41 AM

If you check, the Origami and Rosetta strains are implmented with a plasmid containing genes for the rare codons, so, yes, this can definitely be done.
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).

#5 AaronF

AaronF

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 03 January 2013 - 12:35 PM

Are you wanting to purify in vitro transcribed tRNA or native tRNA? In some cases the modifications don't matter as much (I could get crucified for saying that) and in other cases they are absolutely necessary. In-vitro transcription gives you more tRNA for the amount of time you spend in the purification process. I haven't done the math, but I'm pretty sure it is less expensive as well.

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.

#6 neuron

neuron

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 187 posts
5
Neutral

Posted 03 January 2013 - 08:03 PM

Thanks so much AaronF!! Its a great help. our purpose is to get good quality and good quantity of specific tRNA. And I think for that we need to clone it only. I will check for puc18 vector and try to design an experiment as you suggested. I will look into details of this and then may be I'll bother you againPosted Image. I have done cloning for genes like putting them into pBS, pET vectors, miniprep etc. But for tRNA I need to see how to design primers also. So yes I need to start it from scratch.




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.