Contribution

Further Characterization of BBa_K2137001

This year, Waterloo iGEM further characterized the Cup1 promoter. This is an inducible promoter that can be used in yeast. It can be triggered by adding copper sulfate to the media used to grow the yeast. The team used the part BBa_K2137001 for this characterization, and performed the characterization in the yeast strain W303. The part contains the Cup1 promoter in front of GFP. In all characterization experiments performed this year, yeast was grown in YPD for ~12 hours, then diluted to an OD of 0.1 to a total volume 25 mL of YPD media. Upon dilution to an OD of 0.1, all samples were split in two, with copper added to one and not the other. The time of copper addition was deemed time 0.

Previous Work

Last year, the Waterloo iGEM team created the part BBa_K2137001 and performed preliminary characterization. This year, the team furthered the characterization by examining the effect of varying copper concentration used to induce the promoter and observing the fluorescent cells with a flow cytometer instead of a plate reader (which was used in 2016). The flow cytometer allowed for a more in-depth analysis of the cell population rather than just a net fluorescence reading.

Dual Populations

The first finding of this characterization study was that yeast cells fall into two “populations,” one fluorescent and the other not. Since the yeast was grown in complete media, the non-fluorescent population most likely consisted of cells that had lost the plasmid containing the Cup1 promoter and GFP. The non-fluorescent population grew slowly but steadily over time, but the rate of this growth would most likely vary if growth conditions were altered in any significant way.

Fluorescent Population

For the purposes of the remainder of this study, the term “fluorescent population” will be used to refer to cells with a fluorescence of over 1000 fluorescence units as identified by the flow cytometer when operating a 488 nm laser at a power of 2 mW. The team ran a total of 12 samples of empty yeast cells as controls, and found that these samples never had more than 0.2% of their population above the 1000 fluorescence units threshold.

Results

In order to ease analysis, the data was normalized as follows. The fluorescence of cells with no added copper at time 0 was scaled to 1 for each copper / no copper pair of trials. After this, all other fluorescence values in that trial were scaled by the same factor. The experiment outlined above, performed by adding copper sulfate to YPD until reaching a concentration of 250 um, was performed in triplicate, giving results as follows:

As one can see, at all data points but one the cells induced with copper have higher fluorescence than those in the non-induced sample.

Our additional finding was that when growing yeast in YPD, the increase in the induction of Cup1 plateaus after a copper concentration of about 125 uM.

Samples of empty cells with copper were run to determine that the copper by itself was not responsible for the increased fluorescence.