Disruption of genes in yeast

[ahlou]

hello to everyone i need some help about my research i have been trying to obtain triple mutant in saccharomyces cerevisiae lacking all three pyruvate decarboxylase genes but i cannot get colonies incomplex media having glucose as carbon source..i am using the LiAc/PEG/SS DNA protocol ...till now i am successful to obtain double mutant but not triple mutan...i need really your help because my work depends on that triple mutant..Thanks for your help

[chadn737]

Other Y2H screens I have not worked in Yeast. Has anybody made a triple mutant before? Are you sure its not lethal?

[vampares]

 

i cannot get colonies incomplex media having glucose as carbon source

Pyruvate is a major chemical pathway cross road. Getting back up hill without further metabolism would be difficult.

 

I'm going to be trite and say that once you are here, at pyruvate, turn around and ask yourself why you are here. The answer is probably because gulcose do to the nature of chemical structures only looks like 2 pyruvates. You would have to do something with this at some point in time. Its rly simple.

 

Just grow them on something other than glucose. Ah ha but there in lies the problem.

[CharonY]

Pyruvate is a major chemical pathway cross road. Getting back up hill without further metabolism would be difficult.

 

I'm going to be trite and say that once you are here, at pyruvate, turn around and ask yourself why you are here. The answer is probably because gulcose do to the nature of chemical structures only looks like 2 pyruvates. You would have to do something with this at some point in time. Its rly simple.

 

Just grow them on something other than glucose. Ah ha but there in lies the problem.

Does this make sense to anyone?

 

With regards to OP, naively I would have thought that it is not essential under aerobic conditions. However, the produced acetaldehyde could also be funneled into other pathways which could negatively affect growth. Actually, I faintly recall a paper in which multiple pyruvate decarboxylase mutants were described. It was a Dutch group, but I cannot recall the names right now (must be around the 90s and for some reasons I remember that they were big on chemostats). But if I recall correctly they would grow on complex medium but not minimal with only glucose. Or something to that effect, at least. If OP is still interested I can try to dig it out (have not worked with yeast for a loooong time),

Edited March 23, 2014 by CharonY

[chadn737]

Does this make sense to anyone?

 

Absolutely none. Glad someone else thought the same.