Regulatory

Part:BBa_K431007:Experience

Designed by: David Joseph Sexton Jr.   Group: iGEM10_Georgia_State   (2010-09-17)
Revision as of 16:55, 27 September 2013 by Andresochoa (Talk | contribs) (→‎Applications of BBa_K431007)

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Applications of BBa_K431007

Results

Characterization

Pichia growth on ethanol solutions

To test the promoters response to the inputs, we did quick preliminary tests with P. pastoris to evaluate it survival ability to ethanol concentrations to define a specific ethanol range for input testing.

<img src="USPBrPichiaGrowth.png" style="border: none;" width="600" height="246" />
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.

After making a growth curve of Pichia pastoris on simple YPD media (graph above), we defined the OD of the stationary phase. With this information we tested the yeast growth repression in presence of ethanol, measuring samples in different ethanol concentration solutions when the control sample (without ethanol) achieved the stationary phase. The results are following:

<img src="USPBrPichiaGrowthRepress.png" style="border: none;" width="600" height="246" />
Figure 4

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


4.5% Ethanol
<img src="USPBrPichia45Eth.png" width="200" height="200" />		 6% Ethanol<b/>
<img src="USPBrPichia60Eth.png" width="200" height="200" /></td>

</tr>

<b>7.5% Ethanol
<img src="USPBrPichia75Eth.png" width="200" height="200" />		 9% Ethanol
<img src="USPBrPichia90Eth.png" width="200" height="200" />

Figure 5

This result shows us that, besides stagnation on growth, the cells remain viable. Further tests relating to this subject were done and could be found hereafter on lyophilization results. For now, this information is enough to proceed with the characterization of our planned strains, knowing that the cells resist ethanol percentages normally found in alcoholic beverages.

Measuring Input Intensity Response from Strain Control 1

With the first successful transformation on Pichia pastoris, the Control 1 Strain (see transformation results), we already measured the cells response on a range of concentration of methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters for mRFP1 fluorescence assay present on <a href="https://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below for samples from the same cultivation, starting with a OD 0.1:

<img src="USPBrYPfluorescence.png" style="border: none;" width="600" height="382" />
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 preliminary result indicates that exist an inferior limit of PAOX1 expression induction (equal or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable 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 biosensor

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