Part:BBa_K2137002
Gal1-GFP Transcriptional Fusion
It was necessary for us to choose promoters that expressed the constructs we were cloning into yeast cells. We wanted to characterize promoter expression with GFP before we decided which promoter to use in our system with the CFP-Hsp104 construct. Furthermore, we needed to induce a [PSI+] state by overexpression of Sup35 in the cell by inserting another copy of Sup35 into the system via a plasmid. Overall, we had two plasmids that we wanted to easily induce at any time.
We chose to characterize four main promoters that are commonly found in yeast: Gal1, Adh1, and Cup1.
Expression of the GAL1 gene in S. cerevisiae is strongly repressed by growth on glucose. It is shown that two sites within the GAL1 promoter mediate glucose repression. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC361077/pdf/molcellb00045-0329.pdf)
Characterization
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.
Methods and Materials
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.
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 446
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 70
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1098
| None |