Difference between revisions of "Team:Pittsburgh/Notebook"

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<div class="column full_size">
 
<div class="column full_size">
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<span class="anchor" id="Week1"></span>
 
<span class="anchor" id="Week1"></span>
 
<h1>Week 1: May 23 - May 27</h1>
 
<h1>Week 1: May 23 - May 27</h1>
−
<h2>Wet Lab</h2>
+
    <table>
−
<ul>
+
<tr><td><img src="https://static.igem.org/mediawiki/2016/b/b8/T--Pittsburgh--NotebookWetLab.jpg" alt="Wet Lab"></td>
 +
<td><ul>
 
<li>Training begins</li>
 
<li>Training begins</li>
 
<li>Grow Top 10 <a href="https://2016.igem.org/Team:Pittsburgh/Protocols#competent" target="_blank">competent cells</a>.</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>
+
    </td>
 +
</tr>
 +
        <tr>
 +
<td><img src="https://static.igem.org/mediawiki/2016/e/e0/T--Pittsburgh--NotebookDryLab.jpg" alt="Dry Lab"></td>
 +
    <td>
 
<ul>
 
<ul>
 
<li>Brainstorm genetic circuits for a thallium sensor</li>
 
<li>Brainstorm genetic circuits for a thallium sensor</li>
 
<li>Lab safety training</li>
 
<li>Lab safety training</li>
 
</ul>
 
</ul>
 +
      </td>      </tr>
 +
</table>
 
<a href=https://static.igem.org/mediawiki/2016/b/bc/TeamPittsburghNotebookWeek1.pdf target="_blank">Week 1 Notebook</a><br>
 
<a 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>
 
<a href="#Top">Back to Top</a>
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<h1>Week 5: June 20 - June 26</h1>
 
<h1>Week 5: June 20 - June 26</h1>
 
<h2>Wet Lab</h2>
 
<h2>Wet Lab</h2>
−
<h3>Reporter</h3>
 
−
<ul>
 
−
    <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">Ligate</a> T7 promoter -- amilCP construct to terminator</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>
 
 
<h3>Cell-Free Extract</h3>
 
<h3>Cell-Free Extract</h3>
 
     <ul>
 
     <ul>
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         <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>
 +
<h3>Reporter</h3>
 +
<ul>
 +
    <li><a href="https://2016.igem.org/Team:Pittsburgh/Protocols#ligation" target="_blank">Ligate</a> T7 promoter -- amilCP construct to terminator</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>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<ul>
 
<ul>
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<h1>Week 6: June 27 - July 3</h1>
 
<h1>Week 6: June 27 - July 3</h1>
 
<h2>Wet Lab</h2>
 
<h2>Wet Lab</h2>
 +
<h3>Cell-Free Extract</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>
 
<h3>Reporter</h3>
 
<ul>
 
<ul>
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     <li>Linearized plasmids containing only the promoter and insert (no terminator) do not express protein</li>
 
     <li>Linearized plasmids containing only the promoter and insert (no terminator) do not express protein</li>
 
</ul>
 
</ul>
−
<h3>Cell-Free Extract</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>
 
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<ul>
 
<ul>
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<h1>Week 7: July 5 - July 8</h1>
 
<h1>Week 7: July 5 - July 8</h1>
 
<h2>Wet Lab</h2>
 
<h2>Wet Lab</h2>
−
<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>
 
 
<h3>Cell-Free Extract</h3>
 
<h3>Cell-Free Extract</h3>
 
     <ul>
 
     <ul>
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         <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>
 
     </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>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<ul>
 
<ul>
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<h1>Week 8: July 11 - July 17</h1>
 
<h1>Week 8: July 11 - July 17</h1>
 
<h2>Wet Lab</h2>
 
<h2>Wet Lab</h2>
−
<h3>Reporter</h3>
 
−
<ul>
 
−
    <li>Restart amilCP cloning process</li>
 
−
</ul>
 
 
<h3>Cell-Free Extract</h3>
 
<h3>Cell-Free Extract</h3>
 
     <ul>
 
     <ul>
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         <li>Erbium cleaves the P substrate strand</li>
 
         <li>Erbium cleaves the P substrate strand</li>
 
     </ul>
 
     </ul>
 +
<h3>Reporter</h3>
 +
<ul>
 +
    <li>Restart amilCP cloning process</li>
 +
</ul>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<ul>
 
<ul>
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<h1>Week 9: July 18 - July 22</h1>
 
<h1>Week 9: July 18 - July 22</h1>
 
<h2>Wet Lab</h2>
 
<h2>Wet Lab</h2>
−
<h3>Reporter</h3>
 
−
<ul>
 
−
    <li>Continue amilCP cloning process</li>
 
−
    <li>Clone RBS-T3 RNA polymerase to add into other constructs</li>
 
−
</ul>
 
 
<h3>Cell-Free Extract</h3>
 
<h3>Cell-Free Extract</h3>
 
     <ul>
 
     <ul>
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         <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>
 
     </ul>
 +
<h3>Reporter</h3>
 +
<ul>
 +
    <li>Continue amilCP cloning process</li>
 +
    <li>Clone RBS-T3 RNA polymerase to add into other constructs</li>
 +
</ul>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<ul>
 
<ul>

Revision as of 20:08, 30 July 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 1 Notebook
Back to Top

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 2 Notebook
Back to Top

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 3 Notebook
Back to Top

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 4 Notebook
Back to Top

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 5 Notebook
Back to Top

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 consturcts 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 6 Notebook
Back to Top

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 7 Notebook
Back to Top

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 8 Notebook
Back to Top

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
Week 9 Notebook
Back to Top