Team:Waterloo/Results/PromoterAnalysis
Promoter Analysis
The Gal1-GFP inducible promotor was one of the parts our team characterized this year. The reason we characterized this promoter is used to understand the transcription rates in a normal cell in order to see the metabolic load of expression of additional Hsp104. To characterize Gal1, it was cloned in front of GFP so a simple fluorescence assay could be performed. The Gal1 promotor was induced using 2% galactose and is repressed in the presence of glucose. The biobrick part can be found here.
Strains of yeast with the plasmids were grown to an OD600 of 0.1 in YPGal media in 30 degrees Celsius on a shaker at 200 RPM and then were diluted into media and put into a plate reader. For each sample, cells were normalized 107 cells. Measurements of GFP were taken over a period of 24 hours using a Gemini XPS Microplate Reader. This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.
Figure 1. shows that over time, the yeast cells with Gal1-GFP showed relative fluorescence units of 3.4 times at 6 hours over a control with no promoter and 3.6 times at 27 hours. This data shows the effectiveness of the Gal1 promoter over the baseline. Therefore, the promoter has been effectively characterized as GFP shows the expression levels produced by inducing Gal1 with 2% galactose.
The Cup1-GFP transcriptional fusion inducible promoter was another part characterized this year. This construct is the same as the Hsp104 NSC plasmid except the Gal1,10 promoter was replaced by Cup1 and CFP was replaced by GFP. This was used as a positive control to show the normal retention of our constructs with Cup1 promoters by cells. Cup1 was induced using 200 uM copper sulfate.
To characterize Cup1, strains of yeast with the plasmids were grown to an OD600 of 0.1 in YPGal media in 30 degrees Celsius on a shaker at 200 RPM and then were diluted into media and put into a plate reader. For each sample, cells were normalized 107 cells. Measurements of GFP were taken over a period of 24 hours using a Gemini XPS Microplate Reader. This protocol is based off in-house protocols created by previous Waterloo iGEM members and revised over the years by advisors and experienced users.
Figure 2. shows that over time, the yeast cells with Cup1-GFP showed relative fluorescence units 4 times induction over a control with no promoter at 6 hours and 5 times at 27 hours. Therefore, the promoter has been effectively characterized as GFP shows the expression levels produced by inducing Cup1 with 200 uM copper sulfate.
