Team:FAU Erlangen/Tour42
Generating fluorescent yeast strains
During the course of our project we generated several yeast strains, differing in the way they express YFP. Originally derived from k699 the first strain created was YCYFP, transformed with an YCplac22 with the YFP expression construct. After we had confirmed YFP fluorescence we created a strain with the YFP construct actually inserted into the genome by homologous recombination from YIplac204. In this strain we tested the SCATTER constructs by co-transforming YEplac181 containing the Deacetylase part and pTUM12 containing the TAL domain. These strains were named RT1, RT2, and RT3 (for Rpd and Tale1/2/3).
YFP detection
To test the regulatory constructs we wanted to create a reporter that could be easily confirmed after transforming the yeast cells. Therefore the choice fell on a fluorescent protein which we wanted to screen for via fluorescent microscopy. In the end, due to the lack of YFP filters for fluorescence microscopy in our lab we asked another group for help who happened to have a confocal laser scanning microscope we could use. Using the CLSM with an appropriate YFP filter we were actually able to confirm that the cells expressed YFP and thus go to the second step of downregulating the expression.
While the optical confirmation of YFP fluorescence was a good start, we wanted to quantify the expression levels before and after transformation with the regulatory constructs. To do this, we chose RT-qPCR. By quantifying the level of YFP mRNA in the cells it is possible to directly derive the level of transcriptional activity.
We used Tub1, a housekeeping gene in yeast to standardize the data. Because the YFP construct we used had no intron it was necessary to run a –RT control as well, to find out how much genomic DNA remained in the samples during RNA preparation and to compensate for the additional material amplified in the reaction. The data shows a clear decrease in mRNA levels in the yeast strains transformed with the regulatory constructs.