Difference between revisions of "Team:Pittsburgh/Notebook"

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         float:left;
 
         float:left;
 
     }
 
     }
 +
   
 
      
 
      
 
</style>
 
</style>
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<h1 style="clear:both;">Week 2: May 31 - June 3</h1>
 
<h1 style="clear:both;">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;">
 
<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;">
+
<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>
 
     <li>Test <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">efficiency</a> of competent cells</li>
 
</ul>
 
</ul>
−
     <div class="summary" style="padding:5px">
+
      
 
<h3>Cell-Free Extract</h3>
 
<h3>Cell-Free Extract</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>    
 
          
 
          
 
<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;">
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         <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>
 
     </ul>
 
     </ul>
−
     </div>
+
      
−
<div class="summary" style="padding:5px; ">
+
 
<h3>Toehold Switch</h3>
 
<h3>Toehold Switch</h3>
 
     <ul>
 
     <ul>
 
         <li>Collins triggers activate the switches (both in plasmid form) to express LacZ</li>
 
         <li>Collins triggers activate the switches (both in plasmid form) to express LacZ</li>
 
     </ul>
 
     </ul>
−
    </div>
+
 
−
<div class="summary" style="padding:5px; ">
+
 
<h3>Reporter</h3>
 
<h3>Reporter</h3>
 
<ul>
 
<ul>
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     <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>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px;">
+
 
<h3>Toehold Switch</h3>
 
<h3>Toehold Switch</h3>
 
     <ul>
 
     <ul>
 
         <li>Collins plasmids express LacZ with 25 ng of switch</li>
 
         <li>Collins plasmids express LacZ with 25 ng of switch</li>
 
         <li>DNA oligos trigger Collins switches</li>
 
         <li>DNA oligos trigger Collins switches</li>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px;">
+
 
<h3>Reporter</h3>
 
<h3>Reporter</h3>
 
<ul>
 
<ul>
−
     <li>Identify successful ligations to terminator for amilCP and eGFP consturcts using a <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#agarosegel" target="_blank">gel</a></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>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>Test plasmids in <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#cellfree" target="_blank">cell-free extract</a></li>
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     <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>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px;">
+
 
<h3>DNAzyme</h3>
 
<h3>DNAzyme</h3>
 
     <ul>
 
     <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><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>
 
         <li>Test success of annealing reaction in cell-free extract and with acrylamide gels</li>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px; ">
+
 
<h3>Reporter</h3>
 
<h3>Reporter</h3>
 
<ul>
 
<ul>
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     <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>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px;">
+
 
<h3>DNAzyme</h3>
 
<h3>DNAzyme</h3>
 
     <ul>
 
     <ul>
 
         <li>DNAzyme duplex does not trigger toehold switch</li>
 
         <li>DNAzyme duplex does not trigger toehold switch</li>
 
         <li>Erbium cleaves the P substrate strand</li>
 
         <li>Erbium cleaves the P substrate strand</li>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px; ">
+
 
<h3>Reporter</h3>
 
<h3>Reporter</h3>
 
<ul>
 
<ul>
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     <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>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px;">
+
 
<h3>DNAzyme</h3>
 
<h3>DNAzyme</h3>
 
     <ul>
 
     <ul>
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         <li>Six-hour time course of cleavage does not yield much additional information</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>
 
         <li>Reaction temperature (room temperature versus 37°C) does not produce observable effect on cleavage rates</li>
−
     </ul></div>
+
     </ul>
−
<div class="summary" style="padding:5px; "><h3>Reporter</h3>
+
    <h3>Reporter</h3>
 
<ul>
 
<ul>
 
     <li>Continue amilCP cloning process</li>
 
     <li>Continue amilCP cloning process</li>

Revision as of 15:28, 1 August 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 progress. For a list of our protocols, visit the Protocols page

Contents

Week 1: May 23 - May 27

Wet Lab Dry Lab
  • Brainstorm genetic circuits for a thallium sensor
  • Lab safety training

Week 2: May 31 - June 3

Wet Lab

Cell-Free Extract

Dry Lab
  • Contact museums and summer programs for outreach opportunities
  • Lab safety training

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 Tissue Engineering Camp 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
  • Work on outreach presentation for tissue engineering camp

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 tissue engineering camp
  • 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
  • First presentation at Camp BioE
  • Prepare for UMD Mid-Atlantic Meet-Up
  • Contact PLSG and NEB for sponsorship

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
  • Clone RBS-T3 RNA polymerase to add into other constructs
Dry Lab
  • Presentation at Camp BioE
  • UMD Mid-Atlantic Meet-Up
  • Continue fundraising
  • Discuss systems to model