Difference between revisions of "Part:BBa K431007:Experience"

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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/7/76/USPBrPichiaGrowthRepress.png" style="border: none;" width="600" height="246" /><br /><b>Figure 4</b></p>
 
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/7/76/USPBrPichiaGrowthRepress.png" style="border: none;" width="600" height="246" /><br /><b>Figure 4</b></p>
 
<br></br>
 
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<p>Some of the amples were also grow on YDP plates to qualitatively evaluate their viability:</p>
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<p>Some of the samples were also grow on YDP plates to qualitatively evaluate their viability:</p>
  
  
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</tr>
 
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</table><p class="figure"><b>Figure 5</b> </p>
 
</table><p class="figure"><b>Figure 5</b> </p>
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<br></br>
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<p>This result shows us that, besides stagnation on growth, the cells remain viable. Further tests
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<p>This result shows us that, besides stagnation on growth, the cells remain viable. 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. Beverages with higher percentage of ethanol must be diluted to 5-10%  to be used with this device.</p>
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relating to this subject were done and could be found hereafter on lyophilization results. For
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<br></br>
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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.</p>
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<h4>Measuring Input Intensity Response from Strain Control 1</h4>
 
<h4>Measuring Input Intensity Response from Strain Control 1</h4>
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<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see
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<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain ( P<sub>AOX1</sub>-RFP), we already measured the cells response on a range of concentration of  
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transformation results), we already measured the cells response on a range of concentration of  
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methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters  
 
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 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:</p>
 
for samples from the same cultivation, starting with a OD 0.1:</p>
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<br></br>
 
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrYPfluorescence.png" style="border: none;" width="600" height="382" /><br /><b>Figure 6:</b> 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).</p>
 
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrYPfluorescence.png" style="border: none;" width="600" height="382" /><br /><b>Figure 6:</b> 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).</p>
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<br></br>
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<p>This result indicates the inferior limit of P<sub>AOX1</sub> expression induction is equal to 50 mM of methanol. These data is very important to the comprehension of promoter’s function as part of this biosensor.</p>
  
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<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal
 
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or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter
 
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with great precision. These data is very important to the comprehension of promoter’s
 
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function and validation of our mathematical model.</p>
 
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<p> 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</p>
 
  
  

Latest revision as of 18:11, 27 September 2013

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Please enter how you used this part and how it worked out.

Applications of BBa_K431007

The USP team 2013 used this part as a promoter to create a biosensor for alcoholic drinks (which contain ethanol) tainted by methanol. The reporter gene used was the red fluorescent protein (Part:BBa_E1010). Here are the results that show the limits of the chassis P. pastoris for this purpose and the use of this promoter as a biosensor component of this device. If you want to know more about our project click here.…

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.




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:




Figure 4



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

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

Figure 5



This result shows us that, besides stagnation on growth, the cells remain viable. 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. Beverages with higher percentage of ethanol must be diluted to 5-10% 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 ( PAOX1-RFP), 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 BBa_E1010 information. We obtained the graph below for samples from the same cultivation, 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 the inferior limit of PAOX1 expression induction is equal to 50 mM of methanol. These data is very important to the comprehension of promoter’s function as part of this biosensor.

User Reviews

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