Team:USP-Brazil/Results:Assemblies

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<p style="padding-top: 50px; margin-left: -40px;"><img src="https://static.igem.org/mediawiki/2013/d/d1/TitleUSPBrResults.png" width="117" height="47" alt="Results" /></p>
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<h2>Results</h2>
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<h3>Assemblies and Transformations</h3>
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<p>To develop the BioBricks characterization, we built the following strains:</p>
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<h3>Assemblies</h3>
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/7/7d/RESULTS_-_Strain_Map_%28New2%29.png" width="650" height="615" style="border:none;" /><br /><b>Figure 1:</b> Map of strains that will be use for testing the device. DNA construction images hiding RBS and transcription stop sequences.</p>
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<p>To explore the questions on BioBricks characterization, we built the construction of the following strains:</p>
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/01/RESULTS-Strain_Map.png" width="650" height="615" style="border:none;" /><br /><b>Figure 1:</b> Map of planned test strains. DNA construction images hiding RBS and transcription stop sequences.</p>
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<p>The P<sub>AOX1</sub> strains are combinations of three DNA elements: the P<sub>AOX1</sub> promoter, the RFP reporter gene and the modified Mxr1 transcription factor (aforementioned on <a href="https://2013.igem.org/Team:USP-Brazil/Project">Detecthol)</a>. Thanks to <a href="https://2013.igem.org/Team:USP-Brazil/Sponsors">Life Technologies</a>, the strain B1 and the Mxr1 modified and minimum were synthesized and this enabled us to construct the others strains.</p>
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<p>The P<sub>AOX1</sub> strains are basically different combinations of variants of only three DNA elements: the P<sub>AOX1</sub> promoter, the RFP reporter protein and the modified Mxr1 transcription factor (aforementioned on <a href="https://2013.igem.org/Team:USP-Brazil/Project">Detecthol)</a>. Thanks to <a href="https://2013.igem.org/Team:USP-Brazil/Sponsors">Life Technologies</a>, the strain B1 and the Mxr1 modified and minimum were synthesized and this enabled us to construct the others strains.</p>
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<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we  
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<p>Comparing the fluorescence time delay and intensity between strains Control 1, A1, and B1, we  
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will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub>  
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub>  
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control  
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control  
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strains 2 with A2 and B2 will draw the picture of P<sub>AOX1</sub> promoter behavior with the modified Mxr1  
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strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1  
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transcription factor with the same variables from the last comparison, but also testing if the  
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transcription factor is used. This will also test if the shorter version of Mxr1&#8212;consisting in the sequence for the N-terminal 400 amino-acids from  
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shorter version of Mxr1&#8212;consisting in the sequence for the N-terminal 400 amino-acids from  
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Mxr1&#8212;cited on literature [1] will work properly.</p>
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Mxr1&#8212;cited on literature [2] will work properly.</p>
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<p>To assembly those parts we: used the strain B1 as template to built the strain A1 using PCR (polymerase chain reaction) and restriction enzymes; the control strain 1 was assembly in the pPIC9K plasmid, which was used to transform <i>P. pastoris</i>, allowing to integrate the construction by homologous recombination in the genome; the strains A2, B2 and Control strain 2 are made by transforming <i>P. pastoris</i> with the corresponding P<sub>AOX1</sub>-RFP construction and the Mxr1 plasmid.
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… gel… o que a gente conseguiu construir, o que está na linha de produção... e etc.</p>
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<h3>Transformation</h3>
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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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<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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<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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<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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<p>Some 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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<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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<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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<p>This result shows us that, besides stagnation on growth, the cells remain viable. Further tests
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relating to this subject were done and could be found hereafter on lyophilization results. For
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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>
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<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see
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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
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for mRFP1 fluorescence assay present on <a href="http://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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<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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<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal
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<p>To assembly those parts we: used the strain B1 as template to built the strain A using PCR (polymerase chain reaction) and restriction enzymes; the control strain 1 was assembled in the pPIC9K plasmid, which was used to transform <i>P. pastoris</i>, allowing to integrate the construction by homologous recombination in the genome; the strains B2 and Control strain 2 will be made by transforming <i>P. pastoris</i> with the corresponding P<sub>AOX1</sub>-RFP construction and the Mxr1 plasmid at the same time; the strain C will be built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p>
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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>  
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<p> We are building our last construction, strain C, and we already have transformed into <i>P. pastoris</i> two of our constructs. The last construction and the other transformations are planned to be done next week. This will allow us to continue the characterization phase, that we have already done for the Control strain 1.</p>
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<h4 style="color:grey;">References</h4>
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<p style="color:grey;">
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<p>[1] PK Parua et al. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Molecular Microbology, vol. 85(2): 282-298 (2012). <br></br></p>
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<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results"><i class="icon-circle-arrow-left"></i> Go back to the Overview</a></p>
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<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization">See the Characterization results <i class="icon-circle-arrow-right"></i></a></p>
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Latest revision as of 04:12, 28 September 2013

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Results

Assemblies and Transformations

To develop the BioBricks characterization, we built the following strains:


Figure 1: Map of strains that will be use for testing the device. DNA construction images hiding RBS and transcription stop sequences.

The PAOX1 strains are combinations of three DNA elements: the PAOX1 promoter, the RFP reporter gene and the modified Mxr1 transcription factor (aforementioned on Detecthol). Thanks to Life Technologies, the strain B1 and the Mxr1 modified and minimum were synthesized and this enabled us to construct the others strains.

Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we will be able to check the strength and the response velocity of the device with modified PAOX1 and the efficiency of codon-optimization on RFP for P. pastoris. The comparison between control strain 2 with strain B2 will show the PAOX1 promoter behavior when the modified Mxr1 transcription factor is used. This will also test if the shorter version of Mxr1—consisting in the sequence for the N-terminal 400 amino-acids from Mxr1—cited on literature [1] will work properly.

To assembly those parts we: used the strain B1 as template to built the strain A using PCR (polymerase chain reaction) and restriction enzymes; the control strain 1 was assembled in the pPIC9K plasmid, which was used to transform P. pastoris, allowing to integrate the construction by homologous recombination in the genome; the strains B2 and Control strain 2 will be made by transforming P. pastoris with the corresponding PAOX1-RFP construction and the Mxr1 plasmid at the same time; the strain C will be built using the RFP condon/otimized and the PFLD promoter.

We are building our last construction, strain C, and we already have transformed into P. pastoris two of our constructs. The last construction and the other transformations are planned to be done next week. This will allow us to continue the characterization phase, that we have already done for the Control strain 1.

References

[1] PK Parua et al. Pichia pastoris 14-3-3 regulates transcriptional activity of the methanol inducible transcription factor Mxr1 by direct interaction. Molecular Microbology, vol. 85(2): 282-298 (2012).

Go back to the Overview

See the Characterization results

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