http://2013.igem.org/wiki/index.php?title=Special:Contributions/Andresochoa&feed=atom&limit=50&target=Andresochoa&year=&month=2013.igem.org - User contributions [en]2024-03-29T06:16:10ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:USP-Brazil/EventsTeam:USP-Brazil/Events2013-09-28T04:13:24Z<p>Andresochoa: </p>
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<p style="padding-top: 50px; margin-left: -40px;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices"><img src="https://static.igem.org/mediawiki/2013/e/e8/USPBrazilHumanPractices.png" width="258" height="49" alt="Human Practices" /></a></p><br />
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<h2>Events</h2><br />
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<p>.</p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/4/40/USPBrAndres1.jpg/800px-USPBrAndres1.jpg" width="450" /><br />
<br /><b>Figure 1:</b> Andres Ochoa giving a presentation to high school students.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/7/74/Picture_451.jpg/800px-Picture_451.jpg" width="450" /><br />
<br /><b>Figure 2:</b> Andres Ochoa giving a presentation at a national science meeting.</p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/ce/USPBrAndres3.jpg/800px-USPBrAndres3.jpg" width="450" /><br />
<br /><b>Figure 3:</b> Andres Ochoa giving a presentation at a international science meeting.</p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/e/ee/USPBrAndres5.jpg/800px-USPBrAndres5.jpg" width="450" /><br />
<br /><b>Figure 4:</b> Pedro Medeiros and Andres Ochoa presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a>.</p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/USPBrAndres6.jpg/800px-USPBrAndres6.jpg" width="450" /><br />
<br /><b>Figure 5:</b> Andres presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a></p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/c8/Modelagem_com_o_Felippe_%283%29.jpg/800px-Modelagem_com_o_Felippe_%283%29.jpg" width="450" /><br />
<br /><b>Figure 6:</b> Felippe giving a presentation about models.</p><br />
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<br />
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<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures"><i class="icon-circle-arrow-left"></i> See our Online Lectures</a></p><br />
<p style="float: right;">For more events, check our <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">Collaborations section <i class="icon-circle-arrow-right"></i></a></p><br />
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<p style="text-align:center;"><a href="https://2013.igem.org/Team:USP-Brazil/GeneralOpinion">Public Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/ExpertsOpinion">Specialists Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/CardGame">Synbio Card Game</a> | <a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures">Online Lectures</a> | Events</p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T04:12:08Z<p>Andresochoa: </p>
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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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
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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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [1] will work properly.</p><br />
<br />
<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><br />
<br />
<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> <br />
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<h4 style="color:grey;">References</h4><br />
<p style="color:grey;"><br />
<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><br />
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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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T04:11:35Z<p>Andresochoa: </p>
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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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [1] will work properly.</p><br />
<br />
<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><br />
<br />
<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> <br />
<br />
h4 style="color:grey;">References</h4><br />
<p style="color:grey;"><br />
<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><br />
<br />
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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><br />
<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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/EventsTeam:USP-Brazil/Events2013-09-28T04:05:26Z<p>Andresochoa: </p>
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<p style="padding-top: 50px; margin-left: -40px;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices"><img src="https://static.igem.org/mediawiki/2013/e/e8/USPBrazilHumanPractices.png" width="258" height="49" alt="Human Practices" /></a></p><br />
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<h2>Events</h2><br />
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<p>.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/4/40/USPBrAndres1.jpg/800px-USPBrAndres1.jpg" width="450" /><br />
<br /><b>Figure 1:</b> Andres Ochoa giving a presentation to high school students.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/7/74/Picture_451.jpg/800px-Picture_451.jpg" width="450" /><br />
<br /><b>Figure 2:</b>Andres Ochoa giving a presentation </p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/ce/USPBrAndres3.jpg/800px-USPBrAndres3.jpg" width="450" /><br />
<br /><b>Figure 3:</b>Andres Ochoa giving a presentation</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/e/ee/USPBrAndres5.jpg/800px-USPBrAndres5.jpg" width="450" /><br />
<br /><b>Figure 4:</b> Pedro Medeiros and Andres Ochoa presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a>.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/USPBrAndres6.jpg/800px-USPBrAndres6.jpg" width="450" /><br />
<br /><b>Figure 5:</b> Andres presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a></p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/c8/Modelagem_com_o_Felippe_%283%29.jpg/800px-Modelagem_com_o_Felippe_%283%29.jpg" width="450" /><br />
<br /><b>Figure 6:</b> Felippe giving a presentation about models.</p><br />
<br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures"><i class="icon-circle-arrow-left"></i> See our Online Lectures</a></p><br />
<p style="float: right;">For more events, check our <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">Collaborations section <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
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<p style="text-align:center;"><a href="https://2013.igem.org/Team:USP-Brazil/GeneralOpinion">Public Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/ExpertsOpinion">Specialists Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/CardGame">Synbio Card Game</a> | <a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures">Online Lectures</a> | Events</p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/EventsTeam:USP-Brazil/Events2013-09-28T04:04:32Z<p>Andresochoa: </p>
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<div style="width: 800px; margin: auto; text-style:center;"><br />
<p style="padding-top: 50px; margin-left: -40px;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices"><img src="https://static.igem.org/mediawiki/2013/e/e8/USPBrazilHumanPractices.png" width="258" height="49" alt="Human Practices" /></a></p><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<br />
<h2>Events</h2><br />
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<p>.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/4/40/USPBrAndres1.jpg/800px-USPBrAndres1.jpg" width="450" /><br />
<br /><b>Figure 1:</b> Andres giving a presentation to high school students.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/7/74/Picture_451.jpg/800px-Picture_451.jpg" width="450" /><br />
<br /><b>Figure 2:</b> </p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/ce/USPBrAndres3.jpg/800px-USPBrAndres3.jpg" width="450" /><br />
<br /><b>Figure 3:</b></p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/e/ee/USPBrAndres5.jpg/800px-USPBrAndres5.jpg" width="450" /><br />
<br /><b>Figure 4:</b> Pedro Medeiros and Andres Ochoa presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a>.</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/USPBrAndres6.jpg/800px-USPBrAndres6.jpg" width="450" /><br />
<br /><b>Figure 5:</b> Andres presenting during the <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">event we hosted with other Brasilian iGEM teams</a></p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/thumb/c/c8/Modelagem_com_o_Felippe_%283%29.jpg/800px-Modelagem_com_o_Felippe_%283%29.jpg" width="450" /><br />
<br /><b>Figure 6:</b> Felippe giving a presentation about models.</p><br />
<br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures"><i class="icon-circle-arrow-left"></i> See our Online Lectures</a></p><br />
<p style="float: right;">For more events, check our <a href="https://2013.igem.org/Team:USP-Brazil/Colaborations">Collaborations section <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
<br />
<p style="text-align:center;"><a href="https://2013.igem.org/Team:USP-Brazil/GeneralOpinion">Public Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/ExpertsOpinion">Specialists Opinion</a> | <a href="https://2013.igem.org/Team:USP-Brazil/CardGame">Synbio Card Game</a> | <a href="https://2013.igem.org/Team:USP-Brazil/OnlineLectures">Online Lectures</a> | Events</p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T04:02:19Z<p>Andresochoa: </p>
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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><br />
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<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
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<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
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<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension result after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
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<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water, we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate. In our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours, after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:58:23Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension result after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water, we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate. In our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours, after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:56:52Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension result after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water, we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate, in our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:54:38Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension result after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate, in our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:52:06Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four hours before plating in YPD, which was not much different from the 10% ethanol resuspension after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate, in our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:48:56Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four ours before plating in YPD, which was not much different from the 10% ethanol resuspension after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p>When we normalized the data using the control and express it in percentage, we can observed that after 4 hours resuspension with water we reduced to 40% the quantity of available cells after lyophilizing, which is a good rate, in our case, we will use ethanol between 5 to 10% for the resuspension, so we can expect more than 24% funcional cells until 4 hours after the use of the device (Figure 3).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:43:27Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four ours before plating in YPD, which was not much different from the 10% ethanol resuspension after the same period. In the other hand, the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, showed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:42:52Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four ours before plating in YPD, which was not much different from the 10% ethanol resuspension after the same period. In the other hand the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, shoed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:39:55Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of the same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization process&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>After lyophilization of 10^8 cells, we resuspended and plate immediately or plate four hours after resuspension. We tested water and water-ethanol solutions as resuspension liquid. This allowed us to see a decreased in the number of UFCs from 10^8 to approximately 10^7 when we wait four ours before plating in YPD, which was not much different from the 10% ethanol resuspension for the same period. In the other hand the resuspension with 15% ethanol and 20% ethanol and plating after four hours wait, shoed a decreased to 10^3 and 10^2, respectively (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T03:23:36Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. The use of same methodology as before, UFCs counting, and also concentration of the 1 mL<br />
cultures to 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival rates&#8212; showed very interesting results. </p><br />
<br />
<p>We noticed that four hours after lyophilization, the resuspension with 10% ethanol barely decreased the number of UFCs when compared with the resuspension in water. In the other hand, this same treatment with 15% ethanol decreased to less than 30% the UFCs number when compared with the same period water (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:15:36Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [1] will work properly.</p><br />
<br />
<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><br />
<br />
<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> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:14:16Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [1] will work properly.</p><br />
<br />
<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><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:12:26Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [1] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/ProjectTeam:USP-Brazil/Project2013-09-28T03:08:46Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<html><br />
<br />
<div style="width: 800px; margin: 30px auto 80px auto; text-style:center;"><br />
<p style="padding-top: 50px; margin-left: -40px;"><img src="https://static.igem.org/mediawiki/2013/1/1a/TitleUSPBrDetect.png" width="149" height="49" alt="Detecthol" /></p><br />
<br />
<h4>Overview</h4><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/5/51/USPBrazilDetectholUP.png') no-repeat bottom; width: 800px; min-height: 271px"><br />
<p id="texto" style="margin-left: 257px; padding-top: 51px;">The device consists of a pen made of waterproof material, divided into three compartments, initially isolated one from each-other. Click on the images below to understand how it works.</p><br />
</div><br />
<br />
<br />
<p><img src="https://static.igem.org/mediawiki/2013/0/03/USPBrazilDetectholDOWN.png" width="800" height="293" usemap="projectmap" /><br />
<map name="projectmap"><br />
<area shape="rect" coords="0,0,117,293" href="javascript:$('#texto').html('The first compartment is for collecting the sample. Put the device already squeezed inside the drink and collect the sample by releasing the pressure. After the sample is collected in the first slot, the sampler end should be blent, resulting in the closure of the device.');" <br />
<br />
title="1"><br />
<area shape="rect" coords="214,0,317,293" href="javascript:$('#texto').html('The second compartment is released by pressing the middle-part of the pen, this will put in contact the sample with the GMOs. Then you just need to wait a little.');" <br />
<br />
title="2"><br />
<area shape="rect" coords="370,0,566,293" href="javascript:$('#texto').html('After some time, check if the liquid is red. Is easy to see through, because it is made of transparent plastic. If it is red, it means that there is methanol in the drink and it is activating the yeast expression of the Red Fluorescent Protein (see details in the text below). If it is clear, just relax and enjoy your drink!. <a href=\'https://2013.igem.org/Team:USP-Brazil/Applications\'>For more information, see the Application section</a>.');" <br />
<br />
title="3"><br />
<area shape="rect" coords="646,0,800,293" href="javascript:$('#texto').html('To make everything safer, the last step is to press the upper part of the pen to release the chlorine to eliminate the yeast. <a href=\'https://2013.igem.org/Team:USP-Brazil/Biosafety\'>You can read more about biosafety in our Biosafey page</a>.');" <br />
<br />
title="4"><br />
</map><br />
</p><br />
</div><br />
<br />
<div id="container"><br />
<br />
<h3>The Chassis</h3><br />
<p> Looking for a chassis that would resist the presence of ethanol, and specially of methanol, the simplest solution was to use yeast cells, largely employed in the production of biofuels, such as <i>Saccharomyces cerevisae</i> [6]. Our best choice would be a methylotrophic organism, that is, one that can use methanol as a carbon source [7]. Our chosen yeast, called <i>Pichia pastoris</i>, is a species of methylotrophic yeast, with its genome sequenced [11], that is commonly used in the production of recombinant proteins [8], mainly due to its growth characteristics, such as rapid growth rate and cell density, which make cell suspensions a paste-dense material [9]. It also posses methanol-responsive promoters, that could be part of a genetic circuit that would respond to the presence of methanol. In addition, <i>P.pastoris</i> cultures are able to grow in media with up to 10% of ethanol [10], which makes it a perfect candidate for our chassis. </p><br />
<br />
<br />
<p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/9/9a/SOLUTION_-_Pichia_pastoris.png" width="400"" /><br /><br />
<b>Figure 1:</b> <i>Pichia pastoris</i>, the methylotrophic yeast.<br />
</p><br />
<br />
<br />
<h3>Molecular detection</h3><br />
<br />
<br />
<p>In order to detect methanol in alcoholic drinks, we searched for <i>P.pastoris</i> promoters inducible by methanol, and preferably known to have well-established genetic recombination techniques.</p><br />
<br />
<br />
<p class="table"><br />
<b>Table 1:</b> Best candidate promoters from <i>Pichia pastoris</i> to create our device.<br />
</p><br />
<table><br />
<tr class="tr_first"><td style="width:10%">Promoter name</td><td style="width:35%">Gene product</td><td style="width:35%">Regulation</td><td style="width:20%">Expression level</td></tr><br />
<tr><td>P<sub>AOX1</sub></td><td>Alcohol oxidase promoter 1</td><td>Induced by methanol</td><td>Strong (naturally &sim;5% of mRNA and &sim;30% of total protein)</td></tr><br />
<tr><td>P<sub>AOX2</sub></td><td>Alcohol oxidase promoter 2</td><td>Induced by methanol</td><td>&sim;5%&#8211;10% of P<sub>AOX1</sub></td></tr><br />
<tr><td>P<sub>DAS</sub></td><td>Dihydroxyacetone synthase promoter</td><td>Induced by methanol</td><td>Strong (similar to P<sub>AOX1)</sub></td></tr><br />
<tr><td>P<sub>FLD1</sub></td><td>Formaldehyde dehydrogenase promoter</td><td>Induced by methanol and methylamine</td><td>Strong (similar to P<sub>AOX1</sub>)</td></tr><br />
</table><br />
<br> </br><br />
<br />
<p> The three best choices (see table above) were P<sub>AOX1</sub>, P<sub>FLD1</sub> and P<sub>DAS</sub>. Since we did not find much information on P<sub>DAS</sub>, while P<sub>AOX1</sub> and P<sub>FLD1</sub> are well characterized, both of them having commercial plasmids for genomic integration [13][14], we chose the latter two as our molecular detectors. That, in a simple genetic construction, could work as a proof of concept. <i>A priori</i>, our output system would be the monomeric RFP (Red florescent protein) (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>). The RFP will be use for fluorescence tests and for efficiently characterizing the relative promoter strength. We also expect to be able to see the production of RFP with the naked eye, once in <i>E.coli</i> it is shown to be possible.</p><br />
<br />
<p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/1/15/USPBrasilCultivo.jpg" width="640" height="395" /><br /><br />
<b>Figure 2:</b> Fluorescent proteins expressed in an <i>E. coli</i> suspension. Respectively, amilCP <a href="http://parts.igem.org/Part:BBa_K592009">BBa_K592009</a> (blue), amilGFP <a href="http://parts.igem.org/Part:BBa_K592010">BBa_K592010</a> (yellow) and RFP <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> (red). Image from the Registry of Parts.<br />
</p><br />
<br />
<p> Our team chose to focus on the detection system via methanol-responsive promoters, and to characterize them well as BioBricks. </p><br />
<br />
<br />
<h4>P<sub>AOX1</sub></h4><br />
<p>P<sub>AOX1</sub> is a strong promoter which can be controlled by simple changes in its carbon source [16], and it is the most common choice for expression of heterologous proteins in <i>P. pastoris</i>, having a naturally elevated expression rate, of around 5% of the RNA and 30% of total protein production [12] [15].</p><br />
<br />
<p>The challenge in using P<sub>AOX1</sub> is its regulation. This promoter is prone to a strong catabolic repression by hexoses and ethanol [17] — the main component of alcoholic beverages. Fortunately, ethanol is also involved in the degradation of peroxisomes, cellular compartments where <i>P. pastoris</i> realizes the metabolism of methanol; this aspect is interesting for our application, since it means methanol will not be degraded as fast as it would in the absence of ethanol. Therefore, the degradation of peroxisomes would enhance the activation of P<sub>AOX1</sub>, by allowing methanol to stay for longer in the cell.</p><br />
<br />
<p>In order to develop this biosensor, it is necessary to evaluate the rates at which the promoter P<sub>AOX1</sub> is activated via methanol or inhibited via ethanol. In addition, we decided to study a modification on <i>Pichia pastoris</i> Mxr1 transcription factor [18] that should alter its interaction with P<sub>AOX1</sub> by turning ethanol into an activator of the promoter [19]. If the rate of activation by ethanol stays below the rate of activation by methanol, the latter should be identifiable when the drink is diluted. It would then, be possible to create a color guide that would help one differ pure and contaminated beverages (<a href="https://2013.igem.org/Team:USP-Brazil/Modeling">For more information, see the Modeling section</a>).</p><br />
<br />
<p>In addition to testing the P<sub>AOX1</sub> that is native in <i>Pichia pastoris</i>, we have synthesized a modified version of this promoter with up to 33% more strength than the wild type promoter [20], according to the hypothesis that a stronger promoter should allow better visualization of the colorimetric output at naked eye (<a href="https://2013.igem.org/Team:USP-Brazil/Parts">For more information, see the Parts section</a>).</p><br />
<br />
<h4>P<sub>FLD1</sub></h4><br />
<p>This second promoter has a high transcription rate when activated, just like P<sub>AOX1</sub>, but unlike the previous one is activated by methylamine or methanol [12]. Also, it is not repressed by hexoses, like P<sub>AOX1</sub>. Since an extensive search in scientific literature did not uncover any data on the regulation of P<sub>FLD1</sub> by ethanol, this promoter represents a great alternative to P<sub>AOX1</sub>. If it is confirmed that they do not share the same repression characteristic related to this dicarbonyl alcohol, we could eliminate an issue in our project.</p><br />
<br />
<h4>Mxr1 (methanol expression regulator 1) modified</small></h4><br />
<p>Mxr1 (methanol expression regulator 1) is a key regulator, at transcriptional level, of methanol metabolism in the methylotrophic yeast <i>Pichia pastoris</i>. It is a transcriptional factor that binds upstream of the MUT (methanol utilizing) pathway and peroxisome biogenesis gene´s promoters using its zinc finger domain. Among the MUT genes is the AOX1 gene that is regulated by the P<sub>AOX1</sub> promoter. <br />
<p>The 14-3-3 proteins are ubiquitous, and have important roles in controlling a wide variety of cellular processes, like gene expression, metabolism, cell cycle and apoptosis. These 14-3-3 proteins are involved in the carbon source-dependent regulation of Mxr1, which is inactive in ethanol and glycerol, but is active in methanol.</p><br />
<p>Our submitted Mxr1 is mutated in order to NOT be repressed by ethanol, as the original Mxr1 is. This is done by substituted the Ser215 of the protein with Ala, inactivating the 14-3-3 protein interaction (phosphorylation) with Mxr1. It is also smaller than the original Mxr1 because it was already reported that the major activation domain of Mxr1 is located within the first 400 amino acids.</p><br />
<p>The modified Mxr1 is able to activate the P<sub>AOX1</sub>promoter in ethanol, methanol or in a ethanol/methanol solution. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normally containing 10 to 60% ethanol). This will allow government to make a preliminary high-throughput screening of ethanol drinks tainted will methanol.</p><br />
<br />
<h3>Preservation mechanism</h3><br />
<br />
<p>In order to allow portability and storing of the device, and to create a robust, resistant and fast-responding methanol detector, some lyophilization tests were performed, aiming to produce results similar to <i>Saccharomyces cerevisae</i> yeast granules. That was made possible by adapting some protocols, originally for different yeast species to <i>Pichia pastoris</i>. Some tests were realized, essentially looking for answers to the questions below:</p><br />
<p><ul><br />
<li>How many cells would have to be lyophilized?</li><br />
<li>What is the ideal dilution of the drink?</li><br />
<li>How long would it take between the contact of yeast and the drink to obtain results? </li><br />
</ul></p><br />
<br />
<br />
<br />
<h4>Freeze-dry</h4><br />
<br />
<p>As a strategy to develop a biological system cheap enough for the specific economic issue related to the population involved on the risk of methanol poisoning, our detector should be very cheap. It also should be easy enough to prepare for storage and remain functional through long periods of time. An easy way to do so is the commonly used methodology for long storage of food, biological samples and pharmaceuticals: the lyophilization or freeze-drying process [19]. Maybe one of the most known examples of lyophilized product is the dry yeast, used for baking dough and for other food recipes.</p><br />
<br />
<table><br />
<tr><br />
<td style="width: 580px; border:none;"><p>Just to evaluate how cheap would be to produce a biological detector, we could take&#8212;as an estimative&#8212;the average cost of dry yeast on retail market, which is around 74.54 g per American dollar [20]. So, if the cost to produce and sell the net weight of our detector by lyophilization was the same of the average price in retail market, the user would spend less than five cents by net weight for each unitary detector (approximately 0.04 USD!), considering the use of 3 grams of lyophilized material per product. This is enough to justify the use of a biodetector (and, of course, the lyophilization conservation method) for the economic profile of the population involved in this problem over the world.</p></td><br />
<td style="width: 205px; border:none;"><p class="figure"><img src="https://static.igem.org/mediawiki/2013/5/5a/DETECTHOL_-_Preservation_Mechanism_-_Cost_of_net_weigh_estimative.png" width="200" /><br /><b>Figure 3:</b> 0.04 USD.</p></td><br />
</tr><br />
</table><br />
<br />
<table><br />
<tr><br />
<td style="width: 225px; border:none;"><p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8f/DETECTHOL_-_Preservation_Mechanism_-_Lyophilizator.JPG" width="200" /><br /><br />
<b>Figure 4:</b> Lyophilizator: essentially a vacuum machine.</p></td><br />
<td style="width: 570px; border:none; vertical-align: top;"><p>The lyophilization process is quite simple. It is ased on completely removing water from a sample by sublimation of ice by vacuum [19]. In matter of microbial cultures, the freezing process must be the most instantaneous as possible in order to form little and more dispersed ice crystals, that could lead to lethal or sublethal effects on cells [19][21]. You may check our <i>Pichia</i> lyophilization protocol in our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>.</p></td><br />
</tr><br />
</table><br />
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<p style="text-align:right"><br />
<span style="float:left"><a href="https://2013.igem.org/Team:USP-Brazil/Solution"><i class="icon-circle-arrow-left"></i> Back to the solution</a><br />
</span> <a href="https://2013.igem.org/Team:USP-Brazil/Applications">See the product <i class="icon-circle-arrow-right"></i></a><br />
</p><br />
<br />
<h4 style="color:grey;">References</h4><br />
<p style="color:grey;"><br />
<br />
[1] AJA van Maris et al. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek, vol 90: 391–418 (2006). <br></br><br />
<br />
[2] CP Hollenberg and G Gellissen. Production of recombinant proteins by methylotrophic yeasts. Current Opinion in Biotechnology, vol. 8: 554-560 (1997). <br></br><br />
<br />
[3] LM Damasceno, CJ Huang and CA Batt. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, vol 93:31-39 (2012). <br></br><br />
<br />
[4] JM Cregg et al. Expression in the Yeast Pichia pastoris. Methods in Enzymology, vol. 463, ch. 13 (2009). <br></br><br />
<br />
[5] F Ganske and UT Bornscheuer. Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the inonic liquids [BMIM][BF4] and [BMIM][PF6] and organic solvents. Biotechnology Letters, vol. 28: 465-469 (2006). <br></br><br />
<br />
[6] K De Schutter et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology, vol. 27(6) (2009). <br></br><br />
<br />
[7] T Vogl and A Glieder. Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnology, vol. 30(4): 385-404 (2013). <br></br><br />
<br />
[8] http://www.lifetechnologies.com/order/catalog/product/V23020?ICID==%253Dsearch-product <br></br><br />
<br />
[9] http://www.lifetechnologies.com/order/catalog/product/V17520 <br></br><br />
<br />
[10] https://2010.igem.org/Team:Imperial_College_London/Modules/Fast_Response <br></br><br />
<br />
[11] M Jalic et al. Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastoris. Biotechnol. Prog. vol. 22: 1465-1473 (2006).<br></br><br />
<br />
[12] M Inan and M Meagher. Non-Repressing Carbon Sources for Alcohol Oxidase Promoter of Pichia pastoris. Journal of Bioscience and Bioengineering, vol 92(6): 585-589 (2001).<br></br><br />
<br />
[13] GP Lin-Cereghino et al. Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris. Molecular and Cellular Biology, vol. 26(3): 833-897 (2006).<br></br><br />
<br />
[14] 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><br />
<br />
[15] FS Hartner et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Research, vol 36(12) (2008).<br></br><br />
<br />
[16] S Shen et al. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene, vol. 216: 93-102 (1998). <br></br><br />
<br />
[17] Average price calculated in sep/2013: <br />
https://www.scienceexchange.com/services/gas-chromatography-mass-spectrometry-gc-ms?page=1 <br></br><br />
<br />
[18] http://jenslabs.com/2013/06/06/ketosense-an-arduino-based-ketosis-detector/ <br></br><br />
<br />
[19] JG Day et al. Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, second edition (2007).<br></br><br />
<br />
[20] Average price calculated using 9 different product's costs on USA market (september of 2013)<br />
http://www.amazon.com/s/ref=nb_sb_noss_1/187-4864785-9882854?url=search-alias%3Dgrocery&field-keywords=dry%20yeast&sprefix=dry+ye%2Cgrocery&rh=i%3Agrocery%2Ck%3Adry%20yeast <br></br><br />
<br />
[21] RJ Heckly and J Quay. A Brief Review of Lyophilization Damage and Repair in Bacterial Preparations. Cryobiology, vol. 18: 592-597 (1981).<br></br><br />
<br />
[22] X Polomska et al. Freeze-Drying Preservation of Yeast Adjunct Cultures for Cheese Production. Polish Journal of Food and Nutrition Sciences, vol 62(3): 143-150 (2012).<br></br><br />
<br />
[23] Sreekrishna, Koti, and Keith E. Kropp. "Pichia pastoris." Nonconventional Yeasts in Biotechnology. Springer Berlin Heidelberg, 1996. 203-253.<br></br><br />
<br />
[24] F Ganske and UT Bornscheuer. Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the inonic liquids [BMIM][BF4] and [BMIM][PF6] and organic solvents. Biotechnology Letters, vol. 28: 465-469 (2006).<br></br><br />
<br />
</p><br />
<br />
<br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/ProjectTeam:USP-Brazil/Project2013-09-28T03:07:40Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<html><br />
<br />
<div style="width: 800px; margin: 30px auto 80px auto; text-style:center;"><br />
<p style="padding-top: 50px; margin-left: -40px;"><img src="https://static.igem.org/mediawiki/2013/1/1a/TitleUSPBrDetect.png" width="149" height="49" alt="Detecthol" /></p><br />
<br />
<h4>Overview</h4><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/5/51/USPBrazilDetectholUP.png') no-repeat bottom; width: 800px; min-height: 271px"><br />
<p id="texto" style="margin-left: 257px; padding-top: 51px;">The device consists of a pen made of waterproof material, divided into three compartments, initially isolated one from each-other. Click on the images below to understand how it works.</p><br />
</div><br />
<br />
<br />
<p><img src="https://static.igem.org/mediawiki/2013/0/03/USPBrazilDetectholDOWN.png" width="800" height="293" usemap="projectmap" /><br />
<map name="projectmap"><br />
<area shape="rect" coords="0,0,117,293" href="javascript:$('#texto').html('The first compartment is for collecting the sample. Put the device already squeezed inside the drink and collect the sample by releasing the pressure. After the sample is collected in the first slot, the sampler end should be blent, resulting in the closure of the device.');" <br />
<br />
title="1"><br />
<area shape="rect" coords="214,0,317,293" href="javascript:$('#texto').html('The second compartment is released by pressing the middle-part of the pen, this will put in contact the sample with the GMOs. Then you just need to wait a little.');" <br />
<br />
title="2"><br />
<area shape="rect" coords="370,0,566,293" href="javascript:$('#texto').html('After some time, check if the liquid is red. Is easy to see through, because it is made of transparent plastic. If it is red, it means that there is methanol in the drink and it is activating the yeast expression of the Red Fluorescent Protein (see details in the text below). If it is clear, just relax and enjoy your drink!. <a href=\'https://2013.igem.org/Team:USP-Brazil/Applications\'>For more information, see the Application section</a>.');" <br />
<br />
title="3"><br />
<area shape="rect" coords="646,0,800,293" href="javascript:$('#texto').html('To make everything safer, the last step is to press the upper part of the pen to release the chlorine to eliminate the yeast. <a href=\'https://2013.igem.org/Team:USP-Brazil/Biosafety\'>You can read more about biosafety in our Biosafey page</a>.');" <br />
<br />
title="4"><br />
</map><br />
</p><br />
</div><br />
<br />
<div id="container"><br />
<br />
<h3>The Chassis</h3><br />
<p> Looking for a chassis that would resist the presence of ethanol, and specially of methanol, the simplest solution was to use yeast cells, largely employed in the production of biofuels, such as <i>Saccharomyces cerevisae</i> [6]. Our best choice would be a methylotrophic organism, that is, one that can use methanol as a carbon source [7]. Our chosen yeast, called <i>Pichia pastoris</i>, is a species of methylotrophic yeast, with its genome sequenced [11], that is commonly used in the production of recombinant proteins [8], mainly due to its growth characteristics, such as rapid growth rate and cell density, which make cell suspensions a paste-dense material [9]. It also posses methanol-responsive promoters, that could be part of a genetic circuit that would respond to the presence of methanol. In addition, <i>P.pastoris</i> cultures are able to grow in media with up to 10% of ethanol [10], which makes it a perfect candidate for our chassis. </p><br />
<br />
<br />
<p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/9/9a/SOLUTION_-_Pichia_pastoris.png" width="400"" /><br /><br />
<b>Figure 1:</b> <i>Pichia pastoris</i>, the methylotrophic yeast.<br />
</p><br />
<br />
<br />
<h3>Molecular detection</h3><br />
<br />
<br />
<p>In order to detect methanol in alcoholic drinks, we searched for <i>P.pastoris</i> promoters inducible by methanol, and preferably known to have well-established genetic recombination techniques.</p><br />
<br />
<br />
<p class="table"><br />
<b>Table 1:</b> Best candidate promoters from <i>Pichia pastoris</i> to create our device.<br />
</p><br />
<table><br />
<tr class="tr_first"><td style="width:10%">Promoter name</td><td style="width:35%">Gene product</td><td style="width:35%">Regulation</td><td style="width:20%">Expression level</td></tr><br />
<tr><td>P<sub>AOX1</sub></td><td>Alcohol oxidase promoter 1</td><td>Induced by methanol</td><td>Strong (naturally &sim;5% of mRNA and &sim;30% of total protein)</td></tr><br />
<tr><td>P<sub>AOX2</sub></td><td>Alcohol oxidase promoter 2</td><td>Induced by methanol</td><td>&sim;5%&#8211;10% of P<sub>AOX1</sub></td></tr><br />
<tr><td>P<sub>DAS</sub></td><td>Dihydroxyacetone synthase promoter</td><td>Induced by methanol</td><td>Strong (similar to P<sub>AOX1)</sub></td></tr><br />
<tr><td>P<sub>FLD1</sub></td><td>Formaldehyde dehydrogenase promoter</td><td>Induced by methanol and methylamine</td><td>Strong (similar to P<sub>AOX1</sub>)</td></tr><br />
</table><br />
<br> </br><br />
<br />
<p> The three best choices (see table above) were P<sub>AOX1</sub>, P<sub>FLD1</sub> and P<sub>DAS</sub>. Since we did not find much information on P<sub>DAS</sub>, while P<sub>AOX1</sub> and P<sub>FLD1</sub> are well characterized, both of them having commercial plasmids for genomic integration [13][14], we chose the latter two as our molecular detectors. That, in a simple genetic construction, could work as a proof of concept. <i>A priori</i>, our output system would be the monomeric RFP (Red florescent protein) (<a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a>). The RFP will be use for fluorescence tests and for efficiently characterizing the relative promoter strength. We also expect to be able to see the production of RFP with the naked eye, once in <i>E.coli</i> it is shown to be possible.</p><br />
<br />
<p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/1/15/USPBrasilCultivo.jpg" width="640" height="395" /><br /><br />
<b>Figure 2:</b> Fluorescent proteins expressed in an <i>E. coli</i> suspension. Respectively, amilCP <a href="http://parts.igem.org/Part:BBa_K592009">BBa_K592009</a> (blue), amilGFP <a href="http://parts.igem.org/Part:BBa_K592010">BBa_K592010</a> (yellow) and RFP <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> (red). Image from the Registry of Parts.<br />
</p><br />
<br />
<p> Our team chose to focus on the detection system via methanol-responsive promoters, and to characterize them well as BioBricks. </p><br />
<br />
<br />
<h4>P<sub>AOX1</sub></h4><br />
<p>P<sub>AOX1</sub> is a strong promoter which can be controlled by simple changes in its carbon source [16], and it is the most common choice for expression of heterologous proteins in <i>P. pastoris</i>, having a naturally elevated expression rate, of around 5% of the RNA and 30% of total protein production [12] [15].</p><br />
<br />
<p>The challenge in using P<sub>AOX1</sub> is its regulation. This promoter is prone to a strong catabolic repression by hexoses and ethanol [17] — the main component of alcoholic beverages. Fortunately, ethanol is also involved in the degradation of peroxisomes, cellular compartments where <i>P. pastoris</i> realizes the metabolism of methanol; this aspect is interesting for our application, since it means methanol will not be degraded as fast as it would in the absence of ethanol. Therefore, the degradation of peroxisomes would enhance the activation of P<sub>AOX1</sub>, by allowing methanol to stay for longer in the cell.</p><br />
<br />
<p>In order to develop this biosensor, it is necessary to evaluate the rates at which the promoter P<sub>AOX1</sub> is activated via methanol or inhibited via ethanol. In addition, we decided to study a modification on <i>Pichia pastoris</i> Mxr1 transcription factor [18] that should alter its interaction with P<sub>AOX1</sub> by turning ethanol into an activator of the promoter [19]. If the rate of activation by ethanol stays below the rate of activation by methanol, the latter should be identifiable when the drink is diluted. It would then, be possible to create a color guide that would help one differ pure and contaminated beverages (<a href="https://2013.igem.org/Team:USP-Brazil/Modeling">For more information, see the Modeling section</a>).</p><br />
<br />
<p>In addition to testing the P<sub>AOX1</sub> that is native in <i>Pichia pastoris</i>, we have synthesized a modified version of this promoter with up to 33% more strength than the wild type promoter [20], according to the hypothesis that a stronger promoter should allow better visualization of the colorimetric output at naked eye (<a href="https://2013.igem.org/Team:USP-Brazil/Parts">For more information, see the Parts section</a>).</p><br />
<br />
<h4>P<sub>FLD1</sub></h4><br />
<p>This second promoter has a high transcription rate when activated, just like P<sub>AOX1</sub>, but unlike the previous one is activated by methylamine or methanol [12]. Also, it is not repressed by hexoses, like P<sub>AOX1</sub>. Since an extensive search in scientific literature did not uncover any data on the regulation of P<sub>FLD1</sub> by ethanol, this promoter represents a great alternative to P<sub>AOX1</sub>. If it is confirmed that they do not share the same repression characteristic related to this dicarbonyl alcohol, we could eliminate an issue in our project.</p><br />
<br />
<h4>Mxr1 (methanol expression regulator 1) modified</small></h4><br />
<p>Mxr1 (methanol expression regulator 1) is a key regulator, at transcriptional level, of methanol metabolism in the methylotrophic yeast <i>Pichia pastoris</i>. It is a transcriptional factor that binds upstream of the MUT (methanol utilizing) pathway and peroxisome biogenesis gene´s promoters using its zinc finger domain. Among the MUT genes is the AOX1 gene that is regulated by the P<sub>AOX1</sub> promoter. The P<sub>AOX1</sub> promoter is found three times in the registry of parts; Part:BBa_K431007, Part:BBa_K945000 and BBa_I764001; it is used for heterologous protein expression in Pichia.</p><br />
<p>The 14-3-3 proteins are ubiquitous, and have important roles in controlling a wide variety of cellular processes, like gene expression, metabolism, cell cycle and apoptosis. These 14-3-3 proteins are involved in the carbon source-dependent regulation of Mxr1, which is inactive in ethanol and glycerol, but is active in methanol.</p><br />
<p>Our submitted Mxr1 is mutated in order to NOT be repressed by ethanol, as the original Mxr1 is. This is done by substituted the Ser215 of the protein with Ala, inactivating the 14-3-3 protein interaction (phosphorylation) with Mxr1. It is also smaller than the original Mxr1 because it was already reported that the major activation domain of Mxr1 is located within the first 400 amino acids.</p><br />
<p>The modified Mxr1 is able to activate the P<sub>AOX1</sub>promoter in ethanol, methanol or in a ethanol/methanol solution. This is useful for our biosensor design, which aims to detect levels of methanol above 2% in common alcoholic drinks (normally containing 10 to 60% ethanol). This will allow government to make a preliminary high-throughput screening of ethanol drinks tainted will methanol.</p><br />
<br />
<h3>Preservation mechanism</h3><br />
<br />
<p>In order to allow portability and storing of the device, and to create a robust, resistant and fast-responding methanol detector, some lyophilization tests were performed, aiming to produce results similar to <i>Saccharomyces cerevisae</i> yeast granules. That was made possible by adapting some protocols, originally for different yeast species to <i>Pichia pastoris</i>. Some tests were realized, essentially looking for answers to the questions below:</p><br />
<p><ul><br />
<li>How many cells would have to be lyophilized?</li><br />
<li>What is the ideal dilution of the drink?</li><br />
<li>How long would it take between the contact of yeast and the drink to obtain results? </li><br />
</ul></p><br />
<br />
<br />
<br />
<h4>Freeze-dry</h4><br />
<br />
<p>As a strategy to develop a biological system cheap enough for the specific economic issue related to the population involved on the risk of methanol poisoning, our detector should be very cheap. It also should be easy enough to prepare for storage and remain functional through long periods of time. An easy way to do so is the commonly used methodology for long storage of food, biological samples and pharmaceuticals: the lyophilization or freeze-drying process [19]. Maybe one of the most known examples of lyophilized product is the dry yeast, used for baking dough and for other food recipes.</p><br />
<br />
<table><br />
<tr><br />
<td style="width: 580px; border:none;"><p>Just to evaluate how cheap would be to produce a biological detector, we could take&#8212;as an estimative&#8212;the average cost of dry yeast on retail market, which is around 74.54 g per American dollar [20]. So, if the cost to produce and sell the net weight of our detector by lyophilization was the same of the average price in retail market, the user would spend less than five cents by net weight for each unitary detector (approximately 0.04 USD!), considering the use of 3 grams of lyophilized material per product. This is enough to justify the use of a biodetector (and, of course, the lyophilization conservation method) for the economic profile of the population involved in this problem over the world.</p></td><br />
<td style="width: 205px; border:none;"><p class="figure"><img src="https://static.igem.org/mediawiki/2013/5/5a/DETECTHOL_-_Preservation_Mechanism_-_Cost_of_net_weigh_estimative.png" width="200" /><br /><b>Figure 3:</b> 0.04 USD.</p></td><br />
</tr><br />
</table><br />
<br />
<table><br />
<tr><br />
<td style="width: 225px; border:none;"><p class="figure"><br />
<img src="https://static.igem.org/mediawiki/2013/8/8f/DETECTHOL_-_Preservation_Mechanism_-_Lyophilizator.JPG" width="200" /><br /><br />
<b>Figure 4:</b> Lyophilizator: essentially a vacuum machine.</p></td><br />
<td style="width: 570px; border:none; vertical-align: top;"><p>The lyophilization process is quite simple. It is ased on completely removing water from a sample by sublimation of ice by vacuum [19]. In matter of microbial cultures, the freezing process must be the most instantaneous as possible in order to form little and more dispersed ice crystals, that could lead to lethal or sublethal effects on cells [19][21]. You may check our <i>Pichia</i> lyophilization protocol in our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>.</p></td><br />
</tr><br />
</table><br />
<br />
<br />
<br />
<br />
<br />
<p style="text-align:right"><br />
<span style="float:left"><a href="https://2013.igem.org/Team:USP-Brazil/Solution"><i class="icon-circle-arrow-left"></i> Back to the solution</a><br />
</span> <a href="https://2013.igem.org/Team:USP-Brazil/Applications">See the product <i class="icon-circle-arrow-right"></i></a><br />
</p><br />
<br />
<h4 style="color:grey;">References</h4><br />
<p style="color:grey;"><br />
<br />
[1] AJA van Maris et al. Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status. Antonie Van Leeuwenhoek, vol 90: 391–418 (2006). <br></br><br />
<br />
[2] CP Hollenberg and G Gellissen. Production of recombinant proteins by methylotrophic yeasts. Current Opinion in Biotechnology, vol. 8: 554-560 (1997). <br></br><br />
<br />
[3] LM Damasceno, CJ Huang and CA Batt. Protein secretion in Pichia pastoris and advances in protein production. Appl Microbiol Biotechnol, vol 93:31-39 (2012). <br></br><br />
<br />
[4] JM Cregg et al. Expression in the Yeast Pichia pastoris. Methods in Enzymology, vol. 463, ch. 13 (2009). <br></br><br />
<br />
[5] F Ganske and UT Bornscheuer. Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the inonic liquids [BMIM][BF4] and [BMIM][PF6] and organic solvents. Biotechnology Letters, vol. 28: 465-469 (2006). <br></br><br />
<br />
[6] K De Schutter et al. Genome sequence of the recombinant protein production host Pichia pastoris. Nature Biotechnology, vol. 27(6) (2009). <br></br><br />
<br />
[7] T Vogl and A Glieder. Regulation of Pichia pastoris promoters and its consequences for protein production. New Biotechnology, vol. 30(4): 385-404 (2013). <br></br><br />
<br />
[8] http://www.lifetechnologies.com/order/catalog/product/V23020?ICID==%253Dsearch-product <br></br><br />
<br />
[9] http://www.lifetechnologies.com/order/catalog/product/V17520 <br></br><br />
<br />
[10] https://2010.igem.org/Team:Imperial_College_London/Modules/Fast_Response <br></br><br />
<br />
[11] M Jalic et al. Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastoris. Biotechnol. Prog. vol. 22: 1465-1473 (2006).<br></br><br />
<br />
[12] M Inan and M Meagher. Non-Repressing Carbon Sources for Alcohol Oxidase Promoter of Pichia pastoris. Journal of Bioscience and Bioengineering, vol 92(6): 585-589 (2001).<br></br><br />
<br />
[13] GP Lin-Cereghino et al. Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris. Molecular and Cellular Biology, vol. 26(3): 833-897 (2006).<br></br><br />
<br />
[14] 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><br />
<br />
[15] FS Hartner et al. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Research, vol 36(12) (2008).<br></br><br />
<br />
[16] S Shen et al. A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene, vol. 216: 93-102 (1998). <br></br><br />
<br />
[17] Average price calculated in sep/2013: <br />
https://www.scienceexchange.com/services/gas-chromatography-mass-spectrometry-gc-ms?page=1 <br></br><br />
<br />
[18] http://jenslabs.com/2013/06/06/ketosense-an-arduino-based-ketosis-detector/ <br></br><br />
<br />
[19] JG Day et al. Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology, second edition (2007).<br></br><br />
<br />
[20] Average price calculated using 9 different product's costs on USA market (september of 2013)<br />
http://www.amazon.com/s/ref=nb_sb_noss_1/187-4864785-9882854?url=search-alias%3Dgrocery&field-keywords=dry%20yeast&sprefix=dry+ye%2Cgrocery&rh=i%3Agrocery%2Ck%3Adry%20yeast <br></br><br />
<br />
[21] RJ Heckly and J Quay. A Brief Review of Lyophilization Damage and Repair in Bacterial Preparations. Cryobiology, vol. 18: 592-597 (1981).<br></br><br />
<br />
[22] X Polomska et al. Freeze-Drying Preservation of Yeast Adjunct Cultures for Cheese Production. Polish Journal of Food and Nutrition Sciences, vol 62(3): 143-150 (2012).<br></br><br />
<br />
[23] Sreekrishna, Koti, and Keith E. Kropp. "Pichia pastoris." Nonconventional Yeasts in Biotechnology. Springer Berlin Heidelberg, 1996. 203-253.<br></br><br />
<br />
[24] F Ganske and UT Bornscheuer. Growth of Escherichia coli, Pichia pastoris and Bacillus cereus in the presence of the inonic liquids [BMIM][BF4] and [BMIM][PF6] and organic solvents. Biotechnology Letters, vol. 28: 465-469 (2006).<br></br><br />
<br />
</p><br />
<br />
<br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:06:01Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:05:34Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<p>The P<sub>AOX1</sub> strains are combinations of 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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
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</div><br />
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<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:05:05Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<p>The P<sub>AOX1</sub> strains are combinations 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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
</div><br />
<br />
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</div><br />
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<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:04:15Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:03:15Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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 planned test strains. DNA construction images hiding RBS and transcription stop sequences.</p><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
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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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:02:56Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To explore develop the BioBricks characterization, we built the following strains:</p><br />
<br />
<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 planned test strains. DNA construction images hiding RBS and transcription stop sequences.</p><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T03:02:09Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To explore the questions on BioBricks characterization, we built the construction of the following strains:</p><br />
<br />
<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 planned test strains. DNA construction images hiding RBS and transcription stop sequences.</p><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains: Control 1; Strain A and strain B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with strain B2 will show the P<sub>AOX1</sub> promoter behavior when the modified Mxr1 <br />
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 <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 assembled built using the RFP condon/otimized and the P<sub>FLD</sub> promoter. </p><br />
<br />
<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 continuum the characterization phase, that have already be done for the Control strain 1.</p> <br />
<br />
<br />
<br />
<div class="cf"><br />
<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><br />
<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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:AssembliesTeam:USP-Brazil/Results:Assemblies2013-09-28T02:24:31Z<p>Andresochoa: </p>
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<br />
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Assemblies and Transformations</h3><br />
<p>To explore the questions on BioBricks characterization, we built the construction of the following strains:</p><br />
<br />
<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 planned test strains. DNA construction images hiding RBS and transcription stop sequences.</p><br />
<br />
<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><br />
<br />
<p>Comparing the fluorescence time delay and intensity between strains Control 1, A, and B1, we <br />
will be able to check the strength and the response velocity of the device with modified P<sub>AOX1</sub> <br />
and the efficiency of codon-optimization on RFP for <i>P. pastoris</i>. The comparison between control <br />
strain 2 with B2 will draw the picture of P<sub>AOX1</sub> promoter behavior with the modified Mxr1 <br />
transcription factor on the same variables from the last comparison. This will also test if the <br />
shorter version of Mxr1&#8212;consisting in the sequence for the N-terminal 400 amino-acids from <br />
Mxr1&#8212;cited on literature [2] will work properly.</p><br />
<br />
<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 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 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.</p><br />
<br />
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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><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T02:08:31Z<p>Andresochoa: </p>
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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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br />
<br />
<p style="text-align:center;"><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology as before, UFCs counting, and also concentrating the 1 mL<br />
cultures to 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival&#8212; showed very interesting results. </p><br />
<br />
<p>We noticed that four hours after lyophilization, the resuspension with 10% ethanol barely decreased the number of UFCs when compared with the resuspension in water. In the other hand, this same treatment with 15% ethanol decreased to less than 30% the UFCs number when compared with the same period water (Figure 2).<p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br />
<p style="text-align:center;"><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:58:13Z<p>Andresochoa: </p>
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<br />
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<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology as before, UFCs counting, and also concentrating the 1 mL<br />
cultures to 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival&#8212; showed very interesting results. </p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:56:50Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
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<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology as before, UFCs counting, and also concentrating the 1 mL<br />
cultures to 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival showed very interesting results. </p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:55:05Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology as before, UFCs counting, and also concentrating the 1 mL<br />
cultures to a 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival showed very interesting results </p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
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<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:46:21Z<p>Andresochoa: </p>
<hr />
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<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the graph 3, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeast will be resuspended using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology as before, UFCs counting, and also concentrating the 1 mL<br />
cultures to a 50% of its volume before the lyophilization procedure&#8212;trying to get larger survival showed very interesting results, with a surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:36:36Z<p>Andresochoa: </p>
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<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the next graph, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test this preservation method for our intended application, where the yeats will be using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:30:15Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the results showed that milk + glutamate is a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used didn't have residual glucose quantities. As can be seen on the next graph, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying process.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test the usefulness of this preservation method for the application using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:23:46Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<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><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>Serial dilutions of the resuspended lyophilized samples. Each section at each plate corresponds to an ordered area of three (10 uL) drops from culture with incremental dilutions rates (as could be seen in the Milk + Glutamate plate, the rate of dilution increases from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the result showed itself interesting enough for a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used doesn’t have residual glucose quantities. As can be seen on the next graph, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test the usefulness of this preservation method for the application using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization"><i class="icon-circle-arrow-left"></i> See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives <i class="icon-circle-arrow-right"></i></a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:09:37Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Results</h2><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>We serially diluted the resuspended lyophilized samples: each section of plate corresponds to an ordered area of three (10 uL) drops from culture diluted in different orders (as could be seen in the Milk + Glutamate plate, the order of dilutions is from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the result showed itself interesting enough for a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. The milk used doesn’t have residual glucose quantities. As can be seen on the next graph, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test the usefulness of this preservation method for the application using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization">See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives</a></p><br />
</div><br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T01:06:32Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Results</h2><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilization machine), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>We serially diluted the resuspended lyophilized samples: each section of plate corresponds to an ordered area of three (10 uL) drops from culture diluted in different orders (as could be seen in the Milk + Glutamate plate, the order of dilutions is from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the result showed itself interesting enough for a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolized by this yeast [5], this might not affect P<sub>AOX1</sub> activation, if the powdered milk does not have residual glucose quantities. milk doesn’t have residual glucose quantities. As could be seen on the graph next subsection, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test the usefulness of this preservation method for the application using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> cellular density variation on lyophilization process</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistance after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilized. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
<br />
<div class="cf"><br />
<p style="float: left;"><a href="https://2013.igem.org/Team:USP-Brazil/Results:Characterization">See the Characterization results</a></p><br />
<p style="float: right;"><a href="https://2013.igem.org/Team:USP-Brazil/HumanPractices">Check our Human Practices initiatives</a></p><br />
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{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:FreezeDryTeam:USP-Brazil/Results:FreezeDry2013-09-28T00:18:36Z<p>Andresochoa: </p>
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<h2>Results</h2><br />
<br />
<h3>Lyophilization</h3><br />
<h4>Defining the best cryoprotectants </h4><br />
<p>We didn’t found on literature protocols or references about lyophilization of <i>Pichia pastoris</i>, <br />
but&#8212;as the baker’s dry yeasts may confirm&#8212;the protocols for yeasts are abundant. Using as <br />
reference the known methodologies for <i>Saccharomyces cerevisiae</i> and other yeasts [4], we tested <br />
combinations of two cryoprotectants for <i>Pichia’s lyophilization</i>: powered milk and monosodic <br />
glutamate (see our <a href="https://2013.igem.org/Team:USP-Brazil/Notebook">Notebook</a>).</p><br />
<br />
<p>Doing the lyophilization process on 1.5 mL eppendorfs (tip: latter, we found that using 15 <br />
mL falcon tubes is much better to avoid spilling of the samples on the low pressures of the <br />
lyophilizator), the following results were obtained:</p><br />
<br />
<p class="figure"><table style="text-align:center; width: 100%;"><tr><td style="border: none; width:50%;"><b>YPD</b><br /><img src="https://static.igem.org/mediawiki/2013/b/b5/USPBrDefCryYPD.jpg" width="335" height="310" /></td><td style="border: none; width: 50%;"><b>Glutamate<b/><br /><img src="https://static.igem.org/mediawiki/2013/c/cb/USPBrDefCryGlutamate.jpg" width="335" height="310" /></td></tr><tr><td style="border: none;"><b>Milk</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dd/USPBrDefCryMilk.jpg" width="335" height="310" /></td><td style="border: none;"><b>Milk + Glutamate</b><br /><img src="https://static.igem.org/mediawiki/2013/e/e0/USPBrDefCryMilkGlutamate.jpg" width="335" height="310" /></td></tr></table><br /><b>Figure 1:</b>We serially diluted the resuspended lyophilized samples: each section of plate corresponds to an ordered area of three (10 uL) drops from culture diluted in different orders (as could be seen in the Milk + Glutamate plate, the order of dilutions is from right to left and up to dowm).</p><br />
<br />
<p>Although threalose was not used&#8212; witch is also a good protectant for yeast species [4]&#8212;, the result showed itself interesting enough for a cheaper way to make lyophilized <i>Pichia</i>. Since lactose is not metabolised by this yeast [5], this might not affect P<sub>AOX1</sub> activation if the powdered milk does not have residual glucose quantities. milk doesn’t have residual glucose quantities. As could be seen on the graph next subsection, we achieved a very interesting result for the cells viability after lyophilization, reaching around 94% of viability on immediate resuspension of cells after the freeze-drying.</p><br />
<br />
<h4>Ethanol Resistance after Lyophilization</h4><br />
<p>To test the usefulness of this preservation method for the application using ethanol solution <br />
(alcoholic drinks), we tested the survival of <i>P. pastoris</i> cultures in solutions with different ethanol <br />
concentrations. The ability to survive to ethanol medium even after a stressful lyophilization <br />
process is a determinant characteristic that our chassis must have. Using the same methodology <br />
to count UFCs as before (like the previous image), and concentrating two times the 1 mL<br />
cultures using a table-top centrifuge before the lyophilization procedure&#8212;in order to have a <br />
larger survival population&#8212;we achieved very interesting results, with surprisingly a high survival<br />
percentage of cells after four hours of resuspension, reaching around 40% (see graphs below)!</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/d/de/DETECTHOL_-_Preservation_Mechanism_-Celular_Density_Chart.png" width="503" height="256" style="border:none;" /><br /><br /><b>Figure 2:</b> celular density variation on lyophilization proccess</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/0/0d/DETECTHOL_-_Preservation_Mechanism_-Ethanol_Resistence_after_Lyophilization.png" width="503" height="259" style="border:none;" /><br /><br /><b>Figure 3:</b> relative ethanol resistence after lyophilization. Using the &#8220;Control&#8221; (see graph before) as reference.</p><br />
<br />
<p>We used a non-lyophilized serial diluted (YPD) plate of <i>Pichia</i> culture as a control. The plate <br />
of &#8220;t = 0h&#8221; was the immediately resuspension of free-dried <i>P. pastoris</i> on YPD plate and the <br />
&#8220;t = 4h&#8221; plates were resuspensions 4 hours after the &#8220;t = 0&#8221; resuspension. This was done <br />
to simulate the possible scenario with our detector&#8212;when after some couple hours the output <br />
might be come out. As expected, the survival of <i>Pichia</i> drops critically in a solution of higher <br />
concentration of ethanol than 10% [6]. This also corroborates with the <i>Pichia</i>’s grow curves and <br />
ethanol test plates showed previously. Another test was done, in same conditions, with another<br />
ethanol concentration more closely to 10%, and the result was maintained as image below shows.</p><br />
<br />
<p class="figure"><br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<td style="border: none; width:50%;"><b>H<sub>2</sub>O t=0h</b><br /><img src="https://static.igem.org/mediawiki/2013/9/9a/USPBrH2O_t0.png" width="390" height="293" /></td><br />
<td style="border: none; width: 50%;"><b>H<sub>2</sub>O t=4h<b/><br /><img src="https://static.igem.org/mediawiki/2013/0/0b/USPBrH2O_t4.png" width="390" height="293" /></td><br />
</tr><br />
<tr><br />
<td style="border: none;"><b>Ethanol 10% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/4/44/USPBrEth10_t4.png" width="390" height="293" /></td><br />
<td style="border: none;"><b>Ethanol 12.5% t=4h</b><br /><img src="https://static.igem.org/mediawiki/2013/d/dc/USPBrEth12_t4.png" width="390" height="293" /></td><br />
</tr><br />
</table><br /><b>Figure 4:</b> Mind the difference between the dilutions orders contained on each plate&#8212;notations at the plates centers.</p><br />
<br />
<h3>Storage time</h3><br />
<p>The storage of the biodetector is another crucial aspect of it usefulness. The shelf life of <br />
commercial dry yeasts ranges from six months to one year [2], if stored in proper conditions. So, <br />
to efficiently address the project’s challenge, <i>Pichia pastoris</i> must be capable to do the same. We <br />
resuspended some samples from the first lyophilization of “ethanol survival test” after a couple <br />
of weeks. We again surprisingly achieved very good results, showing no significant variation of <br />
cell viability after 3 and 5 weeks after the “t = 0” resuspension from the first ethanol survival test <br />
(see graph below).</p><br />
<br />
<p class="figure"><img src="https://static.igem.org/mediawiki/2013/f/f6/DETECTHOL_-_Preservation_Mechanism_-Relative_Preservation_by_Lyophilization.png" width="572" height="251" style="border:none;" /><br /><br /><b>Figure 5:</b> Relative percentages to the same control of the first ethanol lyophilization test.</p><br />
<br />
<p>A last test we need to prepare is a successive plating of serial dilutions through time of aliquots from a single resuspension, to evaluate the time dependence viability of cells in solution for comparison with the previous results.</p><br />
<br />
<p>We could say that our chosen chassis is also a very good microorganism for storage when lyophilizated. This corroborates with the initial argument that a biodetector could be a functional and very cheap way to solve many social-economic complex problems, like the detection of contaminated alcoholic drinks from non-commercial beverages.</p><br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:16:06Z<p>Andresochoa: </p>
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<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Results</h2><br />
<br />
<h3>Characterization</h3><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain<br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">(see transformation results)</a>, was used to measure the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same batch, starting with a OD 0.1:</p><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><br />
<br />
<p>This result indicates that it exists an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable us to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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 whole biosensor.</p><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:10:35Z<p>Andresochoa: </p>
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<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain<br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">(see transformation results)</a>, was used to measure the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same batch, starting with a OD 0.1:</p><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><br />
<br />
<p>This result indicates that it exists an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable us to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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 whole biosensor.</p><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:10:10Z<p>Andresochoa: </p>
<hr />
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<br />
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<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain<br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">(see transformation results)</a>, was used to measure the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same batch, starting with a OD 0.1:</p><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><br />
<br />
<p>This result indicates that it exists an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable us to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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 whole biosensor</p><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:07:56Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain<br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">(see transformation results)</a>, was used to measure the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same batch, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:06:05Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain<br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:Assemblies">(see transformation results)</a>, was used to measure the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM (approx. 1%) of methanol , following the parameters for the mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:02:51Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br></br><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:01:14Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br />
<h3>Measuring Input Intensity Response from Strain Control 1</h3><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-28T00:00:13Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. 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.</p><br />
<br />
<h4>Measuring Input Intensity Response from Strain Control 1</h4><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-27T23:56:39Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD that corresponds to the stationary phase.<p><br />
<br />
<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><br />
<br />
<p>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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. For <br />
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><br />
<br />
<h4>Measuring Input Intensity Response from Strain Control 1</h4><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-27T23:53:45Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD of the stationary phase.<p><br />
<br />
<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><br />
<br />
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:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>These results showed us that, besides stagnation on growth, the cells remained viable. Further tests <br />
related to this matter were done, and could be found on the <br />
<br />
<a href="https://2013.igem.org/Team:USP-Brazil/Results:FreezeDry">lyophilization results</a>. For <br />
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><br />
<br />
<h4>Measuring Input Intensity Response from Strain Control 1</h4><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-27T23:46:30Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>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 <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>We began by making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below). This allowed us to find the OD of the stationary phase.<p><br />
<br />
<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><br />
<br />
With that information we tested the yeast growth repression in presence of ethanol, measuring samples in different ethanol concentration solutions when the<br />
control sample (without ethanol) achieved the stationary phase. The results are following:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>This result shows us that, besides stagnation on growth, the cells remain viable. Further tests <br />
relating to this subject were done and could be found hereafter on lyophilization results. For <br />
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><br />
<br />
<h4>Measuring Input Intensity Response from Strain Control 1</h4><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoahttp://2013.igem.org/Team:USP-Brazil/Results:CharacterizationTeam:USP-Brazil/Results:Characterization2013-09-27T23:42:06Z<p>Andresochoa: </p>
<hr />
<div>{{https://2013.igem.org/Team:USP-Brazil/templateUP}}<br />
<br />
<html><br />
<div style="background: url('https://static.igem.org/mediawiki/2013/c/c2/USPBrazilBackground.png') no-repeat top right; min-height: 691px;"><br />
<div id="container"><br />
<div style="background: rgba(255,255,255,0.90); padding-right: 10px;"><br />
<br />
<h2>Characterization</h2><br />
<h4>Chassis characterization, behavior of <i>Pichia pastoris</i> growth on ethanol solutions</h4><br />
<p>To test the promoters response to the inputs, we tested <i>P. pastoris</i> survival ability to different ethanol concentrations, this help us to define a specific ethanol range for input testing.</p><br />
<br />
<p>Making a growth curve of <i>Pichia pastoris</i> on YPD media (graphs below), allowed us to find the OD of the stationary phase.<p><br />
<br />
<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><br />
<br />
With that information we tested the yeast growth repression in presence of ethanol, measuring samples in different ethanol concentration solutions when the<br />
control sample (without ethanol) achieved the stationary phase. The results are following:</p><br />
<br />
<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 />
<p>Some were also grow on YDP plates to qualitatively evaluate their viability:</p><br />
<br />
<br />
<table style="text-align:center; width: 100%;"><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
<tr><br />
<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><br />
<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><br />
</tr><br />
</table><p class="figure"><b>Figure 5</b> </p><br />
<br />
<p>This result shows us that, besides stagnation on growth, the cells remain viable. Further tests <br />
relating to this subject were done and could be found hereafter on lyophilization results. For <br />
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><br />
<br />
<h4>Measuring Input Intensity Response from Strain Control 1</h4><br />
<p>With the first successful transformation on <i>Pichia pastoris</i>, the Control 1 Strain (see <br />
transformation results), we already measured the cells response on a range of concentration of <br />
methanol inputs, starting from 0 to 400 mM of methanol (approx. 1%), following the parameters <br />
for mRFP1 fluorescence assay present on <a href="http://parts.igem.org/Part:BBa_E1010">BBa_E1010</a> information. We obtained the graph below <br />
for samples from the same cultivation, starting with a OD 0.1:</p><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><br />
<br />
<p>This preliminary result indicates that exist an inferior limit of P<sub>AOX1</sub> expression induction (equal <br />
or lower than 50 mM of ethanol). This kind of analysis, when applied to the other strains, will enable to characterize the promoter <br />
with great precision. These data is very important to the comprehension of promoter’s <br />
function and validation of our mathematical model.</p><br />
<br />
<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><br />
<br />
<br />
</div><br />
</div><br />
</div><br />
</html><br />
<br />
{{https://2013.igem.org/Team:USP-Brazil/templateDOWN}}</div>Andresochoa