yeast 2 hybrid

Question 1

is yeast 2 hybrid done in vivo or in vitro?

Answer 1

in vivo (inside the cell)

Question 2

what is the point of the yeast 2 hybrid?

Answer 2

see protein interactions

Question 3

what is the difference between co immunoprecipitation and yeast 2 hybrid? what are the advantages

Answer 3

yeast 2 hybrid: in vivo (more natural environment where proteins interact, more false positives and less false negatives)

co ip: in vitro. putting proteins in foreign environment, which could affect their interactions

Question 4

what exactly happens during yeast 2 hybrid?

Answer 4

Firstly, a transcription factor is broken into two parts – a DNA-binding domain (BD) and a catalytic activation domain (AD)
The DNA-binding domain is fused to a protein of interest called the bait (e.g. an enzyme)
The activation domain is fused to a number of potential binding partners – called the prey (e.g. different ligands)
If the bait and prey interact, the two parts of the transcription factor are reconstituted and activate transcription of a gene
If the bait and prey do not interact, the two parts of the transcription factor remain separate and transcription doesn’t occur

Question 5

how do we engineer yeast cells for the yeast 2 hybrid experiment?

Answer 5

-we add the lac z gene under the control of UAS
-we cut Gal 4 (the transcription factor) into two separate parts, therefore the yeast cell doesn’t have a functional Gal4

Question 6

how does the lac z gene allow us to visually observe the fact that transcription is happening?

Answer 6

-it creates b-galactosidase, which cuts x gal, which turns blue when its cut

Question 7

how do we cut Gal4?

Answer 7

-gal 4 AD
-gal 4 BD

Question 8

what is the reporter gene?

Answer 8

lac z

Question 9

what are the 2 negative controls in our experiment?

Answer 9

-in one of the yeast cells, we only put the plasmid that contains the gene that codes for Gal4-BD and the bait, therefore since we dont have AD and the prey, we dont have a functional transcription factor and no transcription will happen

-in one of the yeast cells, we only put the plasmid that contains the gene that codes for Gal4-AD and the prey, therefore since we dont have BD and the bait, we dont have a functional transcription factor and no transcription will happen

Question 10

what is the positive control in our experiment?

Answer 10

gal4 AD and BD are together (no bait or prey). we have a functional Gal4

Question 11

where do the bait and the prey bind?

Answer 11

bait: BD
prey: AD

Question 12

what do BD and AD stand for?

Answer 12

BD: DNA binding domain
AD: activation domain

Question 13

how are BD/AD created so that they’re fused together?

Answer 13

they’re inserted together onto a plasmid to create a recombinant gene where two proteins are fused together