Struggling with Vector Cloning Project

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annakarenina
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Struggling with Vector Cloning Project

Post by annakarenina » Wed Sep 05, 2007 7:17 pm

Hi,

Im a University student doing a really annoying in silico cloning project (using vector NTI software).

DNA and protein technology isnt really my forte (I'm Rubbish at it!!!!) and I was wondering if someone who knows about these things could tell me if I'm pointed in the right direction.

Ok so I have to:

Design and construct an expression vector based on pGEX-2T
that could be used to express my target protein - FAS2 (arabidopsis thaliana fasciata 2) as a recombinant fusion protein with glutathione S transferase in a bacterial host

Okay now I'm sorry if this is a REALLY stupid question, but my University has pretty much piled this assignment on us without any actual teaching and just the aid of a computer lab manual.

so does this mean that im going to use pGEX-2T as my vector.......and fuse my FAS2 protein with glutathione S transferase. so stick that fusion protein into pGEX-2T - and then stick the entire thing in a bacterial host???


Just want to know if I'm totally lost/a bit dim etc.
Thanks for any help anyone can give me!!!!

Cheers

Anna
I think we ought always to entertain our opinions with some measure of doubt. I shouldn't wish people dogmatically to believe any philosophy, not even mine.
Bertrand Russell

blcr11
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Post by blcr11 » Thu Sep 06, 2007 1:17 am

That's about the size of it.

The expression vector, pGEX-2T, carries the GST gene under the control of a tac promoter for expression in bacterial cells. You need to fuse FAS2 in frame with the C-terminus of the GST gene present in pGEX-2T. You'll need to look at the sequence of pGEX-2T to do that correctly. There probably are at least a small number of restriction sites to use. The pGEX-2T-GST-FAS2 plasmid will then be used to transform a suitable bacterial strain for induction of protein expression. I don't know how far you need to take it, but if you were interested in purifying FAS2, you would have to take the fusion protein and clip it with thrombin and then re-purify the FAS2 (or purify away the GST, whichever is easier).

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