Team:USP-Brazil/Results:Characterization

Characterization

Chassis characterization, behavior of Pichia pastoris growth on ethanol solutions

To test the promoters response to the input (methanol) in a medium rich in ethanol (5 to 60% present in common alcoholic drinks), we tested P. pastoris survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.

We began by making a growth curve of Pichia pastoris on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.

Figure 3: Growth curve of Pichia pastoris. Gray x mark the actual data; colored circles represents the mean and the line is the fitted logistic curve. Both curves represent the same conditions, but starting the measuring with two different initial ODs.

With that information we tested the yeast growth repression in presence of ethanol, measuring samples in different ethanol concentration solutions compare to time that takes to the control sample (without ethanol) to achieve the stationary phase. The results are the following:

Figure 4

Some were also grow on YDP plates to qualitatively evaluate their viability:

4.5% Ethanol	6% Ethanol
7.5% Ethanol	9% Ethanol

Figure 5

These results showed us that, besides stagnation on growth, the cells remained viable. Further tests related to this matter were done, and could be found on the lyophilization results. This information allowed us to proceed with the characterization of our planned strains, knowing that the cells resist ethanol percentages normally found in alcoholic beverages. Beverages with higher percentage of ethanol must be diluted to 5 to10%, to be used with this device.

Measuring Input Intensity Response from Strain Control 1

With the first successful transformation on Pichia pastoris, the Control 1 Strain (see transformation results), was used to measure the cells response on a range of concentration of methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on BBa_E1010 information. We obtained the graph below for samples from the same batch, starting with a OD 0.1:

Figure 6: Measure of fluorescent using different mM concentrations of methanol (M400, M200, M100, M50, M25, M12,5). The curves have already being normalized using the control (not induced strain).

This result indicates that it exists an inferior limit of P_AOX1 expression induction (equal or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable us to characterize the promoter with great precision. These data is very important to the comprehension of promoter’s function and validation of our mathematical model.

At this moment we are developing this experiments with the other strains already built, the results wont be ready for the wiki closure but we expect to present some of them in the jamboree, further results require a couple of weeks more, where we will be able to test our whole biosensor.