Difference between revisions of "Team:Pittsburgh/Notebook"

m
 
(102 intermediate revisions by the same user not shown)
Line 2: Line 2:
 
<html>
 
<html>
  
 
+
<style>
 +
   
 +
    .imgDescription {
 +
        position: absolute;
 +
        bottom:45px;
 +
        left:5px;
 +
        padding-right: 5px;
 +
        font-size: 128%;
 +
        width:150%;
 +
    }
 +
   
 +
    .summary {
 +
        display:inline-block;
 +
        float:left;
 +
    }
 +
   
 +
   
 +
</style>
 +
   
 +
   
 +
<div style="max-width:1000px; margin:0 auto; padding:0px 10px 10px 10px;">
 
<div class="column full_size">
 
<div class="column full_size">
  
<p>Our weekly progress</p>
+
<p>Our weekly activities: experiments, data analysis, and planning. For a list of our protocols, visit the <a href="https://2016.igem.org/Team:Pittsburgh/Protocols" target="_blank"> Protocols</a> page.</p>
  
 
</div>
 
</div>
  
<div class="table">
+
<div class="table column full_size" style="padding-top:0;">
<h1><a name="Top">Contents</a></h1>
+
<h2 style="color=#1c2957; padding-top:0;">Contents</h2>
<ul>
+
<ul class="table">
<li><a href="#Week1">Week 1: May 23 - May 27</a></li>
+
<li><a href="#Week1" class="table">Week 1: May 23 - May 27</a></li>
<li><a href="#Week2">Week 2: May 31 - June 3</a></li>
+
<li><a href="#Week2" class="table">Week 2: May 31 - June 3</a></li>
<li><a href="#Week3">Week 3: June 6 - June 12</a></li>
+
<li><a href="#Week3" class="table">Week 3: June 6 - June 12</a></li>
 +
<li><a href="#Week4" class="table">Week 4: June 13 - June 17</a></li>
 +
<li><a href="#Week5" class="table">Week 5: June 20 - June 26</a></li>
 +
<li><a href="#Week6" class="table">Week 6: June 27 - July 3</a></li>
 +
<li><a href="#Week7" class="table">Week 7: July 5 - July 8</a></li>
 +
<li><a href="#Week8" class="table">Week 8: July 11 - July 17</a></li>
 +
<li><a href="#Week9" class="table">Week 9: July 18 - July 22</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="#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>
  
<div class="content">
+
<div class="notebook column full_size">
<h1><a name="Week1">Week 1: May 23 - May 27</a></h1>
+
<span class="anchor" id="Week1"></span>
<h2>Wet Lab</h2>
+
<h1>Week 1: May 23 - May 27</h1>
<ul>
+
 
 +
<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;">
 +
<ul class="summary" style="padding:5px;">
 
<li>Training begins</li>
 
<li>Training begins</li>
<li>Grow Top 10 competent cells.</li>
+
<li>Grow Top 10 <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">competent cells</a>.</li>
 
</ul>
 
</ul>
<h2>Dry Lab</h2>
+
 
<ul>
+
 
 +
<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>Brainstorm genetic circuits for a thallium sensor</li>
 
<li>Brainstorm genetic circuits for a thallium sensor</li>
<li>Lab safety training</li>
+
    <li><a href="2016.igem.org/Team:Pittsburgh/Safety" target="_blank"> Lab safety training</a></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 5px 5px 5px;"><a class="imgDescription" href="https://static.igem.org/mediawiki/2016/b/bc/TeamPittsburghNotebookWeek1.pdf" target="_blank">Week 1 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
<span style="clear:both;"class="anchor" id="Week2"></span>   
 +
<h1>Week 2: May 31 - June 3</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>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">efficiency</a> of competent cells</li>
 +
</ul>
 +
   
 +
<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>
 +
  </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>Contact museums and summer programs for outreach opportunities</li>
 +
    <li><a href="2016.igem.org/Team:Pittsburgh/Safety" target="_blank">Lab safety training</a></li>
 
</ul>
 
</ul>
<a href=https://static.igem.org/mediawiki/2016/b/bc/TeamPittsburghNotebookWeek1.pdf target="_blank">Week 1 Notebook</a>
+
   
 +
<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/a/a8/TeamPittsburghNotebookWeek2.pdf" target="_blank">Week 2 Notebook</a><br>
 
<a href="#Top">Back to Top</a>
 
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
<span style="clear:both;" class="anchor" id="Week3"></span>
 +
<h1>Week 3: June 6 - June 12</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;">
  
<h1><a name="Week2">Week 2: May 31 - June 3</a></h1>
+
    <div class="summary" style="padding:5px; ">
<h2>Wet Lab</h2>
+
<h3>Reporter</h3>
 
<ul>
 
<ul>
<li>Test efficiency of competent cells</li>
+
    <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#transformations" target="_blank">Transform</a> T7 promoter, amilCP, and terminator</li>
<li>Test cell free extract reaction with T7-GFP plasmid</li>
+
    <li>Begin assembly by <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">ligating</a> linearized T7 promoter and amilCP</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>Contact museums and summer programs for outreach opportunities</li>
 
</ul>
 
</ul>
<h2>Dry Lab</h2>
+
   
 +
<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/b/b3/TeamPittsburghNotebookWeek3.pdf" target="_blank">Week 3 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
<span style="clear:both;" class="anchor" id="Week4"></span>   
 +
<h1>Week 4: June 13 - June 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>Reporter</h3>
 
<ul>
 
<ul>
<li>Contact museums and summer programs for outreach opportunities</li>
+
<li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#transformations" target="_blank">Transform</a> T7-GFP plasmid, lacZ alpha fragment, and eGFP</li>
<li>Lab safety training</li>
+
<li>Send promising T7 promoter -- amilCP ligations to be <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#sequencing" target="_blank">sequenced</a></li>
 +
<li>Perform <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#digest" target="_blank">double digest</a> of T7 promoter and terminator from last week</li>
 +
<li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">Ligate</a> double-digested T7 promoter to new reporters (lacZ and eGFP)</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>TECBio, DiSCoBio, and Camp BioE outreach opportunities set</li>
 
</ul>
 
</ul>
<a href=https://static.igem.org/mediawiki/2016/a/a8/TeamPittsburghNotebookWeek2.pdf target="_blank">Week 2 Notebook</a>
+
   
 +
<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/2016/7/75/TeamPittsburghNotebookWeek4.pdf" target="_blank">Week 4 Notebook</a><br>
 
<a href="#Top">Back to Top</a>
 
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
<span  style="clear:both;" class="anchor" id="Week5"></span>   
 +
<h1>Week 5: June 20 - June 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;">
  
<h1><a name="Week3">Week 3: June 6 - June 12</a></h1>
+
<div class="summary" style="padding:5px; ">
<h2>Wet Lab</h2>
+
<h3>Cell-Free Reactions</h3>
 +
    <ul>
 +
        <li>Reaction volume can be reduced to 5 μL with 5 ng/μL of DNA</li>
 +
    </ul>
 +
   
 +
<h3>Toehold Switch</h3>
 +
    <ul>
 +
        <li>Collins triggers activate the switches (both in plasmid form) to express LacZ</li>
 +
    </ul>
 +
 
 +
<h3>Reporter</h3>
 
<ul>
 
<ul>
<li>Transform T7 promoter, amilCP, and terminator</li>
+
    <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">Ligate</a> T7 promoter -- amilCP construct to terminator</li>
<li>Begin assembly by ligating T7 promoter and amilCP</li>
+
<li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#extraction" target="_blank">Extract</a> successful ligations of T7 promoter to eGFP</li>
 +
    <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">Ligate</a> T7 promoter -- eGFP construct to terminator</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>Reach out to teams to collaborate based on last year's projects</li>
 
</ul>
 
</ul>
<h2>Dry Lab</h2>
+
   
 +
<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/2016/7/7c/TeamPittsburghNotebookWeek5.pdf" target="_blank">Week 5 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
<span style="clear:both;" class="anchor" id="Week6"></span>   
 +
<h1>Week 6: June 27 - July 3</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>Reaction volume can be reduced to 1 μL with 5 ng/μL of DNA</li>
 +
    </ul>
 +
<h3>Toehold Switch</h3>
 +
    <ul>
 +
        <li>Collins plasmids express LacZ with 25 ng of switch</li>
 +
        <li>DNA oligos trigger Collins switches</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 
<ul>
 
<ul>
<li>Contact museums and summer programs for outreach opportunities</li>
+
    <li>Identify successful ligations to terminator for amilCP and eGFP constructs using a <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#agarosegel" target="_blank">gel</a></li>
 +
    <li>Send correct plasmids for <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#sequencing" target="_blank">sequencing</a> for confirmation</li>
 +
    <li>Test plasmids in <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract</a></li>
 +
    <li>amilCP does not produce color in cell-free reaction</li>
 +
    <li>eGFP produces fluorescence comparable to that from the Collins T7-GFP plasmid in cell-free reaction</li>
 +
    <li>Linearized plasmids containing only the promoter and insert (no terminator) do not express protein</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 outreach presentation for <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices" target="_blank"> Camp BioE</a></li>
 
</ul>
 
</ul>
<a href=https://static.igem.org/mediawiki/2016/b/b3/TeamPittsburghNotebookWeek3.pdf target="_blank">Week 3 Notebook</a>
+
   
 +
<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/2016/5/59/T--Pittsburgh--NotebookWeek6.pdf" target="_blank">Week 6 Notebook</a><br>
 
<a href="#Top">Back to Top</a>
 
<a href="#Top">Back to Top</a>
 +
    </div>
 +
 +
<span style="clear:both;" class="anchor" id="Week7"></span>   
 +
<h1>Week 7: July 5 - July 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>Cell-Free Reactions</h3>
 +
    <ul>
 +
        <li>384-well plate requires at least 10 μL of reaction</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#annealing" target="_blank">Anneal</a> PO strand with catalytic strand, both with and without erbium</li>
 +
        <li>Test success of annealing reaction in cell-free extract and with acrylamide gels</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
<ul>
 +
    <li>Sequenced amilCP construct does not contain amilCP</li>
 +
    <li>Unsuccessfully linearize and amplify eGFP construct using <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#pcr" target="_blank">PCR</a></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>Practice outreach presentation for <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices" target="_blank">Camp BioE</a></li>
 +
<li>Develop DNAzymes for other heavy metals</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/d/d7/T--Pittsburgh--NotebookWeek7.pdf" target="_blank">Week 7 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week8"></span>   
 +
<h1>Week 8: July 11 - July 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>Cell-Free Reactions</h3>
 +
    <ul>
 +
        <li>Linear eGFP construct does not produce a stronger signal than its plasmid form</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>DNAzyme duplex does not trigger toehold switch</li>
 +
        <li>Erbium cleaves the P substrate strand</li>
 +
    </ul>
 +
<h3>Reporter</h3>
 +
<ul>
 +
    <li>Restart amilCP cloning process</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>First presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices" target="_blank"> Camp BioE</a></li>
 +
    <li>Prepare for <a href="https://2016.igem.org/Team:Pittsburgh/Collaborations#UMD" target="_blank"> UMD Mid-Atlantic Meet-Up</a></li>
 +
<li>Contact PLSG and NEB for sponsorship</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/cb/T--Pittsburgh--NotebookWeek8.pdf" target="_blank">Week 8 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week9"></span>   
 +
<h1>Week 9: July 18 - July 22</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>Reactions can be diluted by one-half and still produce visible results in two hours</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>dPAGE assay of P substrate cleavage suggests that the DNAzyme works, but results are not definitive</li>
 +
        <li>Six-hour time course of cleavage does not yield much additional information</li>
 +
        <li>Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates</li>
 +
    </ul>
 +
    <h3>Reporter</h3>
 +
<ul>
 +
    <li>Continue amilCP cloning process</li></ul>
 +
    <h3>Amplifier</h3>
 +
    <ul><li>Clone RBS-T3 RNA polymerase to add into other 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>Presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices"> Camp BioEhttps://2016.igem.org/Team:Pittsburgh/Collaborations#UMD</li>
 +
<li><a href="https://2016.igem.org/Team:Pittsburgh/Collaborations#UMD" target="_blank"> UMD Mid-Atlantic Meet-Up</a></li>
 +
<li>Continue fundraising</li>
 +
<li>Discuss systems to 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/2016/7/75/T--Pittsburgh--NotebookWeek9.pdf" target="_blank">Week 9 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week10"></span>   
 +
<h1>Week 10: July 25 - July 31</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>Reactions diluted by one-half produce significantly less protein than undiluted reactions</li>
 +
    </ul>
 +
<h3>DNAzyme</h3>
 +
    <ul>
 +
        <li>Annealing reactions produce hybrid complexes but leave unsequestered substrate strand</li>
 +
        <li>DNAzyme does not cleave P substrate strand</li>       
 +
    </ul>
 +
    <h3>Reporter</h3>
 +
<ul>
 +
    <li>Continue cloning amilCP construct</li>
 +
    <li>Determine sequence of possible <i>lacZ</i> plasmid</li>
 +
    <li>Unsuccessfully grow <i>lacZ</i> from iGEM bacterial stab</li>
 +
    </ul>
 +
    <h3>Amplifier</h3>
 +
    <ul>
 +
    <li>Continue cloning 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>Presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices"> Camp BioE</a></li>
 +
<li>Start modeling toehold kinetics and the economical effects of lead</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/2016/6/64/T--Pittsburgh--NotebookWeek10.pdf" target="_blank">Week 10 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </div>   
 +
   
 +
   
 +
<span style="clear:both;" class="anchor" id="Week11"></span>   
 +
<h1>Week 11: August 1 - August 5</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 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>
 +
    <ul>
 +
        <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>
 +
    <h3>Reporter</h3>
 +
<ul>
 +
    <li>amilCP construct contains CFP, not amilCP</li>
 +
    <li>Contact Collins lab for PT3-GFP construct</li>
 +
    </ul>
 +
    <h3>Amplifier</h3>
 +
    <ul>
 +
    <li>Obtain T3 RNA polymerase gene via amplification</li>
 +
    <li>Ligate PT3 and PT3-RBS into plasmid backbone</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>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>Model population effects of lead without economic layer</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/2016/9/9f/T--Pittsburgh--NotebookWeek11.pdf" target="_blank">Week 11 Notebook</a><br>
 +
<a href="#Top">Back to Top</a>
 +
    </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 class="column half_size">
 
<div class="column half_size">
Line 78: Line 721:
  
 
<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

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

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!