Difference between revisions of "Team:Pittsburgh/Notebook"

 
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<div class="table column full_size" style="padding-top:0;">
 
<div class="table column full_size" style="padding-top:0;">
−
<h2 style="color=rgb(0,0,128); padding-top:0;">Contents</h2>
+
<h2 style="color=#1c2957; padding-top:0;">Contents</h2>
 
<ul class="table">
 
<ul class="table">
 
<li><a href="#Week1" class="table">Week 1: May 23 - May 27</a></li>
 
<li><a href="#Week1" class="table">Week 1: May 23 - May 27</a></li>
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<li><a href="#Week10" class="table">Week 10: July 25 - July 31</a></li>
 
<li><a href="#Week10" class="table">Week 10: July 25 - July 31</a></li>
 
<li><a href="#Week11" class="table">Week 11: August 1 - August 5</a></li>
 
<li><a href="#Week11" class="table">Week 11: August 1 - August 5</a></li>
 +
<li><a href="#Week12" class="table">Week 12: August 8 - August 13</a></li>
 +
<li><a href="#Week13" class="table">Week 13: August 15 - August 20</a></li>
 +
<li><a href="#Week14" class="table">Week 14: August 23 - August 26</a></li>
 +
<li><a href="#Week15" class="table">Week 15: August 29 - September 2</a></li>
 +
<li><a href="#Week16" class="table">Week 16: September 6 - September 9</a></li>
 +
<li><a href="#Week17" class="table">Week 17: September 12 - September 17</a></li>
 +
<li><a href="#Week18" class="table">Week 18: September 19 - September 24</a></li>
 +
<li><a href="#Week19" class="table">Week 19: September 28 - September 30</a></li>
 +
<li><a href="#Week20" class="table">Week 20: October 3 - October 8</a></li>
 +
<li><a href="#Week21" class="table">Week 21: October 10 - October 14</a></li>
 +
<li><a href="#Week22" class="table">Week 22: October 17 - October 19</a></li>
 +
<li><a href="#Week23" class="table">Week 23: October 24 - October 31</a></li>
 +
   
 
</ul>
 
</ul>
 
</div>
 
</div>
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</ul>
 
</ul>
 
      
 
      
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul><li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract reaction</a> with T7-GFP plasmid</li></ul>
 
     <ul><li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract reaction</a> with T7-GFP plasmid</li></ul>
 
   </div>     
 
   </div>     
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<div class="summary" style="padding:5px; ">
 
<div class="summary" style="padding:5px; ">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA</li>
 
         <li>Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA</li>
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<div class="summary" style="padding:5px; ">
 
<div class="summary" style="padding:5px; ">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA</li>
 
         <li>Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA</li>
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<div class="summary" style="padding:5px; ">
 
<div class="summary" style="padding:5px; ">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>384-well plate requires at least 10 μL of reaction</li>
 
         <li>384-well plate requires at least 10 μL of reaction</li>
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<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<div class="summary" style="padding:5px;">
 
<div class="summary" style="padding:5px;">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>Linear eGFP construct does not produce a stronger signal than its plasmid form</li>
 
         <li>Linear eGFP construct does not produce a stronger signal than its plasmid form</li>
Line 266: Line 281:
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<div class="summary" style="padding:5px;">
 
<div class="summary" style="padding:5px;">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>Reactions can be diluted by one-half and still produce visible results in two hours</li>
 
         <li>Reactions can be diluted by one-half and still produce visible results in two hours</li>
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<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<div class="summary" style="padding:5px;">
 
<div class="summary" style="padding:5px;">
−
<h3>Cell-Free Extract</h3>
+
<h3>Cell-Free Reactions</h3>
 
     <ul>
 
     <ul>
 
         <li>Reactions diluted by one-half produce significantly less protein than undiluted reactions</li>
 
         <li>Reactions diluted by one-half produce significantly less protein than undiluted reactions</li>
Line 313: Line 328:
 
<ul>
 
<ul>
 
     <li>Continue cloning amilCP construct</li>
 
     <li>Continue cloning amilCP construct</li>
−
     <li>Determine sequence of possible lacZ plasmid</li>
+
     <li>Determine sequence of possible <i>lacZ</i> plasmid</li>
−
     <li>Unsuccessfully grow lacZ from iGEM bacterial stab</li>
+
     <li>Unsuccessfully grow <i>lacZ</i> from iGEM bacterial stab</li>
 
     </ul>
 
     </ul>
 
     <h3>Amplifier</h3>
 
     <h3>Amplifier</h3>
Line 337: Line 352:
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 
<div class="summary" style="padding:5px;">
 
<div class="summary" style="padding:5px;">
 +
    <ul>
 +
    <li>Obtain fluorescence readings for <a href="/Team:Pittsburgh/Collaborations#UGA" target="_blank" >UGA Archaeal InterLab</a></li></ul>
 +
<h3>Cell-Free Reactions</h3>
 +
    <ul>
 +
    <li>Presence of erbium and Buffer B as part of cleavage reaction does not seem to affect  reaction progress</li>
 +
    </ul>
 
<h3>DNAzyme</h3>
 
<h3>DNAzyme</h3>
 
     <ul>
 
     <ul>
−
         <li></li>      
+
         <li>Higher catalytic-to-substrate strand ratios help increase sequestration of substrate strand, especially for G switch</li>
 +
        <li>Cell-free reactions suggest that cleavage of the P substrate strand does occur in the presence of erbium</li>
 +
        <li>dPAGE assay does not suggest cleavage</li>
 
     </ul>
 
     </ul>
 
     <h3>Reporter</h3>
 
     <h3>Reporter</h3>
 
<ul>
 
<ul>
−
     <li></li>
+
     <li>amilCP construct contains CFP, not amilCP</li>
 +
    <li>Contact Collins lab for PT3-GFP construct</li>
 
     </ul>
 
     </ul>
 
     <h3>Amplifier</h3>
 
     <h3>Amplifier</h3>
 
     <ul>
 
     <ul>
−
     <li></li></ul>
+
     <li>Obtain T3 RNA polymerase gene via amplification</li>
 +
    <li>Ligate PT3 and PT3-RBS into plasmid backbone</li>
 +
    </ul>
 
     </div>
 
     </div>
 
      
 
      
Line 354: Line 380:
 
     <li>Presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices"> Camp BioE</a></li>
 
     <li>Presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices"> Camp BioE</a></li>
 
<li>Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science</li>
 
<li>Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science</li>
 +
    <li>Model population effects of lead without economic layer</li>
 
</ul>
 
</ul>
 
      
 
      
 
<div style="position:relative; padding:5px;display:block;float:left;clear:both;">   
 
<div style="position:relative; padding:5px;display:block;float:left;clear:both;">   
−
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="" target="_blank">Week 11 Notebook</a><br>
+
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/9/9f/T--Pittsburgh--NotebookWeek11.pdf" target="_blank">Week 11 Notebook</a><br>
 
<a href="#Top">Back to Top</a>
 
<a href="#Top">Back to Top</a>
 
     </div>     
 
     </div>     
  
 +
   
 +
<span style="clear:both;" class="anchor" id="Week12"></span>   
 +
<h1>Week 12: August 8 - August 13</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Begin work with lead DNAzyme</li>
 +
        <li>dPAGE assay does not suggest cleavage occurs under the current conditions</li>
 +
    </ul>
 +
    <h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagensis of <i>lacZ</i> to remove EcoRI site for BioBricking</li>
 +
    </ul>
 +
    <h3>Amplifier</h3>
 +
    <ul>
 +
    <li>Successfully ligate PT3-RBS into plasmid backbone</li>
 +
    <li>Clone PT3-RBS-T3 and PT7-RBS-T3</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li> <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices">Table at Carnegie Science Center's H<sub>2</sub>Oh! Exhibit</a></li>
 +
    <li>Work on Experiment.com video for crowdfunding</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/5/57/T--Pittsburgh--NotebookWeek12.pdf" target="_blank">Week 12 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
   
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week13"></span>   
 +
<h1>Week 13: August 15 - August 20</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
    <ul>
 +
        <li>Complete data collection for <a href="/Team:Pittsburgh/InterLab" target="_blank"> InterLab study</a></li>
 +
        <li>Prepare William & Mary plasmids for cell-free expression</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Start working with hairpin lead DNAzyme</li>
 +
        <li>Cell-free reactions suggest that a hairpin DNAzyme cleaves more efficiently than a DNAzyme in a duplex</li>
 +
        <li>Adding the DNAzyme, substrate, and metal to a cell-free reaction produces greater switch activation<br> than adding a completed cleavage reaction</li>
 +
        <li>Cell-free reactions suggest that cleavage efficiency is similar at 37°C and room temperature</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i> with DMSO </li>
 +
    </ul>
 +
<h3>Amplifier</h3>
 +
    <ul>
 +
    <li>Unsuccessfully ligate terminator onto PT3-T3 and PT7-T3 constructs</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Work on Experiment.com video for crowdfunding</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/8/8a/T--Pittsburgh--NotebookWeek13.pdf" target="_blank">Week 13 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>   
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week14"></span>   
 +
<h1>Week 14: August 23 - August 26</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
    <ul>
 +
        <li>Complete data collection for William & Mary (read our report <a href="https://static.igem.org/mediawiki/2016/4/46/T--Pittsburgh--CollaborationsWM_report.pdf" target="_blank">here</a>) </li>
 +
    </ul>
 +
<h3>Amplifier</h3>
 +
    <ul>
 +
    <li>ligate terminator onto PT3-T3 and PT7-T3 constructs</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Meet with Dr. Troesken from Pitt's Department of Economics to discuss lead population model</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/2/23/T--Pittsburgh--NotebookWeek14.pdf" target="_blank">Week 14 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>       
 +
   
 +
<span style="clear:both;" class="anchor" id="Week15"></span>   
 +
<h1>Week 15: August 29 - September 2</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>Cell-Free Reactions</h3>
 +
    <ul>
 +
        <li>No reactions, including the positive control, turn purple to indicate the presence of LacZ</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i></li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Move lab up to campus</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/f/f7/T--Pittsburgh--NotebookWeek15.pdf" target="_blank">Week 15 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>     
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week16"></span>   
 +
<h1>Week 16: September 6 - September 9</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
    <ul>
 +
        <li>Obtain codon-optimized OFP, GFP, BFP, YFP, and RFP from 2015 Carnegie Mellon team</li>
 +
    </ul>
 +
<h3>Cell-Free Reactions</h3>
 +
    <ul>
 +
        <li>Plasmids from last week's reactions did not actually contain the toehold switch</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Replicate results from Week 13 suggesting that hairpin lead DNAzyme sequesters and cleaves as expected</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Experiment.com campaign approved</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/7/79/T--Pittsburgh--NotebookWeek16.pdf" target="_blank">Week 16 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>       
 +
   
 +
<span style="clear:both;" class="anchor" id="Week17"></span>   
 +
<h1>Week 17: September 12 - September 17</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme</li>
 +
        <li>Presence of lead may produce signal beyond background activation at 50 nM and 100 nM DNAzyme</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Experiment.com campaign is live</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/4/4d/T--Pittsburgh--NotebookWeek17.pdf" target="_blank">Week 17 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
   
 +
 +
<span style="clear:both;" class="anchor" id="Week18"></span>   
 +
<h1>Week 18: September 19 - September 24</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme</li>
 +
        <li>Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i></li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Modeling</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/5/5a/T--Pittsburgh--NotebookWeek18.pdf" target="_blank">Week 18 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
   
 +
<span style="clear:both;" class="anchor" id="Week19"></span>   
 +
<h1>Week 19: September 28 - September 30</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>PAGE analysis suggests cleavage of hairpin but is not clear</li>
 +
        <li>Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i></li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Modeling</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/4/48/T--Pittsburgh--NotebookWeek19.pdf" target="_blank">Week 19 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
 
 +
   
 +
<span style="clear:both;" class="anchor" id="Week20"></span>   
 +
<h1>Week 20: October 3 - October 8</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>PAGE analysis suggests cleavage of hairpin but is not clear</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i></li>
 +
    </ul>
 +
    </div>
 +
   
 +
<!--<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Modeling</li>
 +
</ul>-->
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/6/62/T--Pittsburgh--NotebookWeek20.pdf" target="_blank">Week 19 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week21"></span>   
 +
<h1>Week 21: October 10 - October 14</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<ul>
 +
    <li>Characterize codon-optimized fluorescent proteins from 2015 Carnegie Mellon team</li>
 +
    </ul>
 +
<h3>Cell-Free Reactions</h3>
 +
    <ul>
 +
    <li>Expected dosage response to lead not observed</li>
 +
    <li>Expected lack of activation with D switch not observed</li>
 +
    <li>Reaction colors do not look the same as in previous weeks</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
    <ul>
 +
    <li>Unsuccessful mutagenesis of <i>lacZ</i></li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Modeling</li>
 +
    <li>Finalize Wiki</li>
 +
</ul>
 +
   
 +
<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/c/cc/T--Pittsburgh--NotebookWeek21.pdf" target="_blank">Week 21 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
 +
 +
    <span style="clear:both;" class="anchor" id="Week22"></span>   
 +
<h1>Week 22: October 17 - October 19</h1>
 +
<img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab" style="padding:5px; width:100px;height:100px;float:left;">
 +
<div class="summary" style="padding:5px;">
 +
<ul>
 +
  <li>Finish cloning Parts </li>
 +
    <li>Submit Parts</li>
 +
    </ul>
 +
    </div>
 +
   
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Finalize Wiki</li>
 +
</ul>
 +
   
 +
    <div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/f/f2/T--Pittsburgh--NotebookWeek22.pdf" target="_blank">Week 22 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div> 
 +
 +
   
 +
   
 +
    <span style="clear:both;" class="anchor" id="Week23"></span>   
 +
<h1>Week 23: October 24 - October 31</h1>
 +
 +
<img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab" style="clear:both; padding:5px; width:100px;height:100px;float:left;">
 +
<ul class="summary" style="padding:5px;">
 +
    <li>Prepare poster and presentation</li>
 +
    <li>Giant Jamboree!</li>
 +
</ul>
 +
    <div style="clear:both;">
 +
    <a href="#Top">Back to Top</a></div>
 +
   
 +
<!--<div style="position:relative; padding:5px;display:block;float:left;clear:both;"> 
 +
<img src="https://static.igem.org/mediawiki/2016/9/96/T--Pittsburgh--NotebookNotebook.png" alt="notebook" style="width:125px;height:auto;padding:0 0 5px 0;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/c/cc/T--Pittsburgh--NotebookWeek21.pdf" target="_blank">Week 21 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>  -->
 +
 +
   
 +
   
 
     </div></div>
 
     </div></div>
  
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<ul>  
 
<ul>  
 +
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 +
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
 +
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
 +
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 +
</ul>
 +
 +
</div>
 +
 +
</html>
 
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
 
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
 
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>

Latest revision as of 20:34, 5 November 2016

Thank you to our sponsors!

Office of the Provost Department of Bioengineering Department of Biological Sciences Department of Chemistry Swanson School of Engineering
IDT NEB Experiment

Our weekly activities: experiments, data analysis, and planning. For a list of our protocols, visit the Protocols page.

Contents

Week 1: May 23 - May 27

Wet Lab Dry Lab

Week 2: May 31 - June 3

Wet Lab

Cell-Free Reactions

Dry Lab

Week 3: June 6 - June 12

Wet Lab

Reporter

  • Transform T7 promoter, amilCP, and terminator
  • Begin assembly by ligating linearized T7 promoter and amilCP
Dry Lab
  • Contact museums and summer programs for outreach opportunities

Week 4: June 13 - June 17

Wet Lab

Reporter

  • Transform T7-GFP plasmid, lacZ alpha fragment, and eGFP
  • Send promising T7 promoter -- amilCP ligations to be sequenced
  • Perform double digest of T7 promoter and terminator from last week
  • Ligate double-digested T7 promoter to new reporters (lacZ and eGFP)
Dry Lab
  • TECBio, DiSCoBio, and Camp BioE outreach opportunities set

Week 5: June 20 - June 26

Wet Lab

Cell-Free Reactions

  • Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA

Toehold Switch

  • Collins triggers activate the switches (both in plasmid form) to express LacZ

Reporter

  • Ligate T7 promoter -- amilCP construct to terminator
  • Extract successful ligations of T7 promoter to eGFP
  • Ligate T7 promoter -- eGFP construct to terminator
Dry Lab
  • Reach out to teams to collaborate based on last year's projects

Week 6: June 27 - July 3

Wet Lab

Cell-Free Reactions

  • Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA

Toehold Switch

  • Collins plasmids express LacZ with 25 ng of switch
  • DNA oligos trigger Collins switches

Reporter

  • Identify successful ligations to terminator for amilCP and eGFP constructs using a gel
  • Send correct plasmids for sequencing for confirmation
  • Test plasmids in cell-free extract
  • amilCP does not produce color in cell-free reaction
  • eGFP produces fluorescence comparable to that from the Collins T7-GFP plasmid in cell-free reaction
  • Linearized plasmids containing only the promoter and insert (no terminator) do not express protein
Dry Lab

Week 7: July 5 - July 8

Wet Lab

Cell-Free Reactions

  • 384-well plate requires at least 10 μL of reaction

DNAzyme

  • Anneal PO strand with catalytic strand, both with and without erbium
  • Test success of annealing reaction in cell-free extract and with acrylamide gels

Reporter

  • Sequenced amilCP construct does not contain amilCP
  • Unsuccessfully linearize and amplify eGFP construct using PCR
Dry Lab
  • Practice outreach presentation for Camp BioE
  • Develop DNAzymes for other heavy metals

Week 8: July 11 - July 17

Wet Lab

Cell-Free Reactions

  • Linear eGFP construct does not produce a stronger signal than its plasmid form

DNAzyme

  • DNAzyme duplex does not trigger toehold switch
  • Erbium cleaves the P substrate strand

Reporter

  • Restart amilCP cloning process
Dry Lab

Week 9: July 18 - July 22

Wet Lab

Cell-Free Reactions

  • Reactions can be diluted by one-half and still produce visible results in two hours

DNAzyme

  • dPAGE assay of P substrate cleavage suggests that the DNAzyme works, but results are not definitive
  • Six-hour time course of cleavage does not yield much additional information
  • Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates

Reporter

  • Continue amilCP cloning process

Amplifier

  • Clone RBS-T3 RNA polymerase to add into other constructs
Dry Lab

Week 10: July 25 - July 31

Wet Lab

Cell-Free Reactions

  • Reactions diluted by one-half produce significantly less protein than undiluted reactions

DNAzyme

  • Annealing reactions produce hybrid complexes but leave unsequestered substrate strand
  • DNAzyme does not cleave P substrate strand

Reporter

  • Continue cloning amilCP construct
  • Determine sequence of possible lacZ plasmid
  • Unsuccessfully grow lacZ from iGEM bacterial stab

Amplifier

  • Continue cloning T3 constructs
Dry Lab
  • Presentation at Camp BioE
  • Start modeling toehold kinetics and the economical effects of lead

Week 11: August 1 - August 5

Wet Lab

Cell-Free Reactions

  • Presence of erbium and Buffer B as part of cleavage reaction does not seem to affect reaction progress

DNAzyme

  • Higher catalytic-to-substrate strand ratios help increase sequestration of substrate strand, especially for G switch
  • Cell-free reactions suggest that cleavage of the P substrate strand does occur in the presence of erbium
  • dPAGE assay does not suggest cleavage

Reporter

  • amilCP construct contains CFP, not amilCP
  • Contact Collins lab for PT3-GFP construct

Amplifier

  • Obtain T3 RNA polymerase gene via amplification
  • Ligate PT3 and PT3-RBS into plasmid backbone
Dry Lab
  • Presentation at Camp BioE
  • Meeting with Dr. Daniel Bain from the University of Pittsburgh Department of Geology and Environmental Science
  • Model population effects of lead without economic layer

Week 12: August 8 - August 13

Wet Lab

DNAzyme

  • Begin work with lead DNAzyme
  • dPAGE assay does not suggest cleavage occurs under the current conditions

Reporter

  • Unsuccessful mutagensis of lacZ to remove EcoRI site for BioBricking

Amplifier

  • Successfully ligate PT3-RBS into plasmid backbone
  • Clone PT3-RBS-T3 and PT7-RBS-T3
Dry Lab

Week 13: August 15 - August 20

Wet Lab
  • Complete data collection for InterLab study
  • Prepare William & Mary plasmids for cell-free expression

DNAzyme

  • Start working with hairpin lead DNAzyme
  • Cell-free reactions suggest that a hairpin DNAzyme cleaves more efficiently than a DNAzyme in a duplex
  • Adding the DNAzyme, substrate, and metal to a cell-free reaction produces greater switch activation
    than adding a completed cleavage reaction
  • Cell-free reactions suggest that cleavage efficiency is similar at 37°C and room temperature

Reporter

  • Unsuccessful mutagenesis of lacZ with DMSO

Amplifier

  • Unsuccessfully ligate terminator onto PT3-T3 and PT7-T3 constructs
Dry Lab
  • Work on Experiment.com video for crowdfunding

Week 14: August 23 - August 26

Wet Lab
  • Complete data collection for William & Mary (read our report here)

Amplifier

  • ligate terminator onto PT3-T3 and PT7-T3 constructs
Dry Lab
  • Meet with Dr. Troesken from Pitt's Department of Economics to discuss lead population model

Week 15: August 29 - September 2

Wet Lab

Cell-Free Reactions

  • No reactions, including the positive control, turn purple to indicate the presence of LacZ

Reporter

  • Unsuccessful mutagenesis of lacZ
Dry Lab
  • Move lab up to campus

Week 16: September 6 - September 9

Wet Lab
  • Obtain codon-optimized OFP, GFP, BFP, YFP, and RFP from 2015 Carnegie Mellon team

Cell-Free Reactions

  • Plasmids from last week's reactions did not actually contain the toehold switch

DNAzyme

  • Replicate results from Week 13 suggesting that hairpin lead DNAzyme sequesters and cleaves as expected
Dry Lab
  • Experiment.com campaign approved

Week 17: September 12 - September 17

Wet Lab

DNAzyme

  • Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme
  • Presence of lead may produce signal beyond background activation at 50 nM and 100 nM DNAzyme
Dry Lab
  • Experiment.com campaign is live

Week 18: September 19 - September 24

Wet Lab

DNAzyme

  • Presence of lead produces signal higher than background hairpin activation at 3.74 nM DNAzyme
  • Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme

Reporter

  • Unsuccessful mutagenesis of lacZ
Dry Lab
  • Modeling

Week 19: September 28 - September 30

Wet Lab

DNAzyme

  • PAGE analysis suggests cleavage of hairpin but is not clear
  • Presence of lead does not produce signal beyond background activation at 50 nM and 100 nM DNAzyme

Reporter

  • Unsuccessful mutagenesis of lacZ
Dry Lab
  • Modeling

Week 20: October 3 - October 8

Wet Lab

DNAzyme

  • PAGE analysis suggests cleavage of hairpin but is not clear

Reporter

  • Unsuccessful mutagenesis of lacZ

Week 21: October 10 - October 14

Wet Lab
  • Characterize codon-optimized fluorescent proteins from 2015 Carnegie Mellon team

Cell-Free Reactions

  • Expected dosage response to lead not observed
  • Expected lack of activation with D switch not observed
  • Reaction colors do not look the same as in previous weeks

Reporter

  • Unsuccessful mutagenesis of lacZ
Dry Lab
  • Modeling
  • Finalize Wiki

Week 22: October 17 - October 19

Wet Lab
  • Finish cloning Parts
  • Submit Parts
Dry Lab
  • Finalize Wiki

Week 23: October 24 - October 31

Dry Lab
  • Prepare poster and presentation
  • Giant Jamboree!