Hi,
I engineered the yeast Saccharomyces cerevisiae to over-express some of its native genes, in addition to native copy of gene. For overexpression, I used strong modified GAL1 promoter ( that is also IPTG-inducible promoter). I would like to study the transcriptional level of the modified genes. To this end, I have to use galactose as a carbon source for yeast cells to grow (because the engineered genes are expressed in the presence of IPTG and galactose, and glucose suppresses the expression). Therefore the yeast cells are growing slow. I inoculated the cells with OD= 0.1 in the induction medium (containing galactose and IPTG). The cells were harvested after reaching OD = 1. It takes the cell around 7 hours to reach this OD.
After performing qPCR, I can not see significant increasement in the expression level of the genes in my samples (containing two copies of each gene, one under the control of the native promoter of gene and one under the control of IPTG inducible promoter) in comparison with control strain that just has the native genes (even the expression level in my samples is less).
Does anyone have experience about right timing (e.g. exponential, or stationary phases) and optimal OD to harvest the yeast cells growing in IPTG/galactose medium to achieve optimal mRNA level? Or what else should I consider to improve the output?
Thanks in advance!
Gita
Hello, mRNA usually expressed in the stationary phase, which was detectable due to the addition of autoinducer molecules in the exponential phase. The better to isolate in stationary phase. OD should be at least 0.8 better above 1.0 at 600nm.
Karen A. Darbinyan Hi Karen,
Thanks for your response! At the moment, I tried OD 1.0,. So based on your comment, it makes sense if I harvest the cultures in higher ODs, too. I will do.
Yes as higher as better buy in stationary phase OD more or less stabile. In the end of stationary phase you can see some little drop on OD due to agregation and precipitation of biomass.
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