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HELP! Try to find genes that can rescue lethality


arscenregi
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Hey guys,

so i´m supposed to develop a method which could help detect genes that can replace an essential gene in yeast by overexpressing them. Does anyone have any ideas as to how i should go about that. i need to figure this out until tumorrow lol, and would appreciate any ideas.

 

thanks in advance

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An overexpression screen? You have a WT yeast, in which you knockout an essential gene, now this has become a lethal phenotype, if during this time you now add start overexpression of a random gene, and your yeast survives, then you have rescued said phenotype. 
One way to do this is to make a overexpression library, get your plate/plates with knockouts and just add the library. Then pick the clones that survive and genotype them (or if you added barcodes to your plasmid library, identify by barcodes). 

If the essential gene knockout leads to a lethal phenotype only under certain conditions (cold sensitivity, some amino acid or other substance auxotrophicity) it is easier, because if your essential gene kills the yeast in all conditions, it is much harder to make sure your initial plate has 100% knockouts (since they won't grow).

hope something like this helps 

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