Galactose induction of Yeast cells
Amita Misra
Friday, 06 February 2009 18:21 UTC
Hello All,
These days I am trying to express my genes in yeast dual expression vector. I have cloned two genes under GAL1 and GAL10 promoters of yeast expression vector. I have transformed competent yeast cells with the recombinant vector and got colonies of yeast in glucose containing medium. But when I tranferred the colonies from glucose to galactose containing medium for expression of my genes under GAL promoters I did not get any growth(16-72hrs of induction time). I have used controls like vector only and one gene at a time also but still I am not getting growth even in controls. I am using 2% final conc. of glucose and galactose.
Plzzz suggest me how to proceed…..
Take care
Amita
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Replies
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The fact that you are not getting any growth in galactose even for your vector controls suggests that something is wrong with the medium. Are you sure all the required aa are available in the medium?
If you are, then it might be that they are being diluted too much from glucose to galactose. Try the induction starting at higher OD’s like 0.5-0.6. Usually you get a delay in growth(about 3hrs) when cells are transferred from glucose to galactose but they should definitely be growing by 16hrs.
You can try adding 1% Raffinose to the Galactose medium to boost growth. Or Go from 2% Glu to 2% raffinose to 2%galactose 1%Raffinose medium. Again make sure you start cultures atleast OD 0.2.
Hope that Helps!
Good Luck
Vinaya -
When I transfer my yeast from glucose to galactose I give them 5 days. Also, before transferring, colony PCR using gene specific primers is a must.
Sometimes after transformation with vector containing GAL1 promoter I just plate them straight on galactose (no glucose step) and they grow.
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Thanks Vinaya for your reply.
Regarding the composition of media I am using YNB and Synthetic URA drop out media from Sigma.
I will try for rafifinose containing medium also. -
Thanks you so much Manojlo for your response.
I have also tried for 3-4 days growth period but nothing worked till now.
I did colony PCR with GAL primers and it is coming good.
I am using 2% final conc. of galactose for induction, is it ok or shall I need to change it?
Saccharomyces strain I am using is YPH499 provided in the kit.
Can you plz tell me the protocol that you use for transformation and induction.That would be a great help for me.
take care,
Amita
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