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

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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===Applications of BBa_K431007===
 
===Applications of BBa_K431007===
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<html>
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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 <a href="https://parts.igem.org/Part:BBa_E1010">(Part:BBa_E1010)</a>. Here are the results that show the limits of the chassis <i>P. pastoris</i> for this purpose and the use of this promoter as a biosensor component of this device. <a href="http://2013.igem.org/Team:USP-Brazil/Project">If you want to know more about our project click here.&#8230;</a>
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<h2>Results</h2>
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<h3>Characterization</h3>
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<h4><i>Pichia</i> growth on ethanol solutions</h4>
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<p>To test the promoters response to the inputs, we did quick preliminary tests with <i>P. pastoris</i> to
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evaluate it survival ability to ethanol concentrations to define a specific ethanol range for input
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testing.</p>
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<br></br>
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/c/c8/USPBrPichiaGrowth.png" style="border: none;" width="600" height="246" /><br /><b>Figure 3:</b> Growth curve of <i>Pichia pastoris</i>. 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.</p>
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<br></br>
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<p>After making a growth curve of <i>Pichia pastoris</i> on simple YPD media (graph above), we defined
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the OD of the stationary phase. With this information we tested the yeast growth repression in
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presence of ethanol, measuring samples in different ethanol concentration solutions when the
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control sample (without ethanol) achieved the stationary phase. The results are following:</p>
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<br></br>
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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>
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<br></br>
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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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<table style="text-align:center; width: 100%;">
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<tr>
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<td style="border: none; width:50%;"><b>4.5% Ethanol</b><br /><img src="https://static.igem.org/mediawiki/2013/0/00/USPBrPichia45Eth.png" width="200" height="200" /></td>
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<td style="border: none; width: 50%;"><b>6% Ethanol<b/><br /><img src="https://static.igem.org/mediawiki/2013/9/9d/USPBrPichia60Eth.png" width="200" height="200" /></td>
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</tr>
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<tr>
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<td style="border: none;"><b>7.5% Ethanol</b><br /><img src="https://static.igem.org/mediawiki/2013/5/50/USPBrPichia75Eth.png" width="200" height="200" /></td>
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<td style="border: none;"><b>9% Ethanol</b><br /><img src="https://static.igem.org/mediawiki/2013/5/5e/USPBrPichia90Eth.png" width="200" height="200" /></td>
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</tr>
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</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. 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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<br></br>
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<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 ( P<sub>AOX1</sub>-RFP), 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
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for mRFP1 fluorescence assay present on <a href="https://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below
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for samples from the same cultivation, starting with a OD 0.1:</p>
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<br></br>
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<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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===User Reviews===
 
===User Reviews===

Latest revision as of 18:11, 27 September 2013

This experience page is provided so that any user may enter their experience using this part.
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

UNIQe532a01a767dfb57-partinfo-00000001-QINU UNIQe532a01a767dfb57-partinfo-00000002-QINU