Difference between revisions of "Part:BBa K2137001"

Latest revision as of 00:14, 22 October 2019

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)

Update (Waterloo iGEM 2019): The original characterization experiments for the promoter were limited by several design flaws. In the original experiments, cells were grown in YPGal media, which is a complex medium. Because of this, it is possible that there was copper in the medium, which would have induced the promoter and prevented us from obtaining accurate characterization data for the promoter. The YPGal media was also not selective for cells containing the vector, meaning that the vector may have been lost in a portion of the cells used for characterization. Finally, the experiments characterized the promoter using only a copper ion concentration of 200 µM.

Characterization

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

Figure 2: OD-corrected Fluorescence vs. Copper Concentration

Figure 3: OD-corrected Fluorescence vs. Copper Concentration (Zoomed)

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

Update (Waterloo iGEM 2019): This year, to improve the characterization of this part, we sought to obtain characterization data for the promoter after growing the cells in a defined medium, WO. This way, we could be sure there was no copper in the growth medium, and that any promoter activity was due to the added copper. This defined media also allowed for selection of cells containing the vector, ensuring that our characterization experiments did not include cells that had lost the vector. Finally, we chose to characterize the promoter by induction with a range of copper concentrations.

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.

Update (Waterloo iGEM 2019): W303a yeast (1) were transformed with a plasmid containing the BBa_K2137001 part, a Cup1-GFP transcriptional fusion. The transformation was completed using standard protocols (2). Following transformation, cells were maintained in selective media before using the Synergy 4 BioTek plate reader to quantify expression.

Using the plate reader, we measured absorbance at 600 nm and GFP fluorescence (ex: 485 nm; em: 525 nm) in 100 µL of sample in a 96 well plate with clear bottoms and black walls. The measurements were taken 8 hours after the addition of varying amounts of copper. The absorbance and GFP fluorescence readings were corrected by subtracting the average values obtained from readings of LB-only blanks. The readings at each copper concentration were taken in triplicate from culture triplicates. The OD-corrected fluorescence was calculated by dividing the GFP fluorescence by the absorbance at 600 nm. The OD-corrected fluorescence of untransformed yeast is 1.080E3 +/- 5.905E2

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.

Update (Waterloo iGEM 2019): Figure 2 displays the OD-corrected fluorescence of the cells containing the Cup1-GFP fusion 8 hours after the addition of varying amounts of copper. These data represent a more robust set of experimental data characterizing the induction of the Cup1 promoter by addition of copper.

Figure 3 contains the same data as figure 2, but allows for better visualization of the data at low copper concentrations.

References

[1] Ralser, M., Kuhl, H., Ralser, M., Werber, M., Lehrach, H., Breitenbach, M., and Bernd, T. The Saccharomyces cerevisiae W303-K6001 cross-platform genome sequence: insights into ancestry and physiology of a laboratory mutt. Open Biol., 2012, 2, 120093.

[2] Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res., 1992, 20, 1425.

Sequence and Features