Part:BBa_K2137001

CUP1-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.

The CUP1 promoter is inducible by adding copper to the medium (http://labs.biology.ucsd.edu/subramani/documents/121.pdf)

Characterization

Figure 1: Relative Fluorescence over time of Cup1-GFP fusion

We performed a series of experiments to test the strength of the promoter using the Cup1-GFP fusion

Methods and Materials

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.

Results and Discussion

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.

Figure 1. 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 292
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 941