Difference between revisions of "Team:Pittsburgh/Notebook"

Line 39: Line 39:
 
<li><a href="#Week8" class="table">Week 8: July 11 - July 17</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="#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>
 
</ul>
 
</ul>
 
</div>
 
</div>
Line 56: Line 58:
 
<ul class="summary" style="padding:5px;">
 
<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>
 
</ul>
  
Line 79: Line 81:
 
<ul class="summary" style="padding:5px; ">
 
<ul class="summary" style="padding:5px; ">
 
<li>Contact museums and summer programs for outreach opportunities</li>
 
<li>Contact museums and summer programs for outreach opportunities</li>
<li>Lab safety training</li>
+
    <li><a href="2016.igem.org/Team:Pittsburgh/Safety" target="_blank">Lab safety training</a></li>
 
</ul>
 
</ul>
 
      
 
      
Line 124: Line 126:
 
<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;">
 
<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;">
 
<ul class="summary" style="padding:5px;">
<li>TECBio, DiSCoBio, and Tissue Engineering Camp outreach opportunities set</li>
+
<li>TECBio, DiSCoBio, and Camp BioE outreach opportunities set</li>
 
</ul>
 
</ul>
 
      
 
      
Line 190: Line 192:
 
<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;">
 
<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; ">
 
<ul class="summary" style="padding:5px; ">
<li>Work on outreach presentation for tissue engineering camp</li>
+
<li>Work on outreach presentation for <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices" target="_blank"> Camp BioE</a></li>
 
</ul>
 
</ul>
 
      
 
      
Line 220: Line 222:
 
<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;">
 
<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;">
 
<ul class="summary" style="padding:5px;">
<li>Practice outreach presentation for tissue engineering camp</li>
+
    <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>
 
<li>Develop DNAzymes for other heavy metals</li>
 
</ul>
 
</ul>
Line 249: Line 251:
 
<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;">
 
<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;">
 
<ul class="summary" style="padding:5px;">
<li>First presentation at Camp BioE</li>
+
    <li>First presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices" target="_blank"> Camp BioE</a></li>
<li>Prepare for UMD Mid-Atlantic Meet-Up</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>
 
<li>Contact PLSG and NEB for sponsorship</li>
 
</ul>
 
</ul>
Line 276: Line 278:
 
     <h3>Reporter</h3>
 
     <h3>Reporter</h3>
 
<ul>
 
<ul>
     <li>Continue amilCP cloning process</li>
+
     <li>Continue amilCP cloning process</li></ul>
     <li>Clone RBS-T3 RNA polymerase to add into other constructs</li>
+
     <h3>Amplifier</h3>
 +
    <ul><li>Clone RBS-T3 RNA polymerase to add into other constructs</li>
 
     </ul></div>
 
     </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;">
 
<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;">
 
<ul class="summary" style="padding:5px;">
<li>Presentation at Camp BioE</li>
+
<li>Presentation at <a href="https://2016.igem.org/Team:Pittsburgh/Human_Practices"> Camp BioEhttps://2016.igem.org/Team:Pittsburgh/Collaborations#UMD</li>
<li>UMD Mid-Atlantic Meet-Up</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>Continue fundraising</li>
 
<li>Discuss systems to model</li>
 
<li>Discuss systems to model</li>
Line 292: Line 295:
 
<a href="#Top">Back to Top</a>
 
<a href="#Top">Back to Top</a>
 
     </div>
 
     </div>
 +
   
 +
   
 +
<span class="anchor" id="Week10"></span>   
 +
<h1 style="clear:both;">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 Extract</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 lacZ plasmid</li>
 +
    <li>Unsuccessfully grow lacZ 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>   
 +
 
     </div></div>
 
     </div></div>
  

Revision as of 01:49, 2 August 2016

Our weekly progress. 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 Extract

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 Extract

  • 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 Extract

  • 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 Extract

  • 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 Extract

  • 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 Extract

  • 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 Extract

  • 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