two hybrid system
pagel at abraxas.magic.lan
Fri Mar 24 23:18:31 EST 2000
> The woman who is teaching me is doing a two hybrid system to screen for
> protein interactions. The yeast strain she uses is AH109 (-ade, -his,
> -lac z). She explained these mutations are the reporter genes. If you
> grow yeast that has been transformed with two plasmids (one a bait
> plasmid and one a library protein plasmid) and grow on LT DO plates,
> then that means the transformation worked.
AH109 is auxotrophic for leucine (L) and tryptophan (W). Therfore, you
know that your transforation worked when you have colonies on SD-W,L
Since you say you are doing a library screen (in contrast to a directed
two-hybrid assay) you plate on SD-W,L only to check for transformation
effieciency, while the major fraction of your transformation is spread on
plates that select for the reporter genes in addition: SD-W,L,H,A
All clones that grow on those plates potentially contain an interacting
fusion-protein from the library.
> She then
> said the next step is a plasmid rescue (the library protein plasmid),
> foll by KC8 transformation.
Depending on the system and the number of positives you have the next step
is to rescue the library plasmid e.g. transforming KC8 with DNA isolate
from the yeast clones.
> Next you isolate the bacterial plasmid, do
> a miniprep and "retransform" the yeast (that has the bait plasmid in it)
> to see if there is a true interaction.
Yes - you want to rule out that your library fusion-protein activates the
transcription of the reporter gene by itself.
> MY question is: why do you need to plate out these "retransformations"
> on 4 different media plates (LT, ALT, HLT, AHLT). It seems to me that
> if you have growth on AHLT that means that a) the yeast successfully
> integrated the library protein AND turned on ade and trp. So why the
> other plates?
First of all I don't think you will leave out the tryptophan, because that
is usually the selection for the bait plasmid (at least in the system I'm
using). If you drop out tryptophane you will probably not get a single
colony, because your yeast doesn't have a bait plasmid.
That leaves us with the following plates:
At the first glance you are right: growth on plate 4 means that the
transformation worked and both reporter genes are active.
But what does it tell you if the yeast does NOT grow on those plates?
- No activation of reporter genes (that's what you hope for)
- No activation of ONE of the reporter genes
- Transformation did not work
Plate 1 tells you that your transformation worked. plates 2 and 3 tell you
basically the same as 4, but with lower stringency.
Hope it helps
Dr. Philipp Pagel
Cellular and Molecular Physiology phone: (203) 785-6835
333 Cedar ST
New Haven, CT 06520
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