Difference between revisions of "Team:WashU StLouis/Notebook"

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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 68 </p>
+
<ul>
 +
<li>Started induction but had to stop bc no frozen stock of <i>fldA</i>-<i>pfo</i> or <i>petF</i>-<i>fldA</i> in DH10B</li>
 +
<li>Transformed <i>fldA</i>-<i>pfo</i> and <i>petF</i>-<i>pfo</i> into DH10B</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 7th</div>
 
     <div class="text">August 7th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 69 </p>
+
<ul>
 +
<li>Made frozen stock of 𝛥<i>aceE</i></li>
 +
<li>Made 60% ethanol</li>
 +
<li>Incubated 8 constructs for biotin assay w/ methanol extraction (4 constructs and blank in DH10B and 𝛥<i>aceE</i>)</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 8th</div>
 
     <div class="text">August 8th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 70 </p>
+
<ul>
 +
<li>Methanol extraction assay (grew cells and froze pellets)</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 9th</div>
 
     <div class="text">August 9th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 71 </p>
+
<ul>
 +
<li>Assayed <i>petF</i>-<i>aceE</i> cells. Absorbance was too low to be meaningful</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 10th</div>
 
     <div class="text">August 10th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 72 </p>
+
<ul>
 +
<li>Ran preliminary sonication test</li>
 +
<li>Incubated large volume of DH10B</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 11th</div>
 
     <div class="text">August 11th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 73 </p>
+
<ul>
 +
<li>Ran large sonication test on DH10B to decide protocol</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 12th</div>
 
     <div class="text">August 12th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 74 </p>
+
<ul>
 +
<li>Incubated large volume of DH10B</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 13th</div>
 
     <div class="text">August 13th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 75 </p>
+
<ul>
 +
<li>More sonication tests</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 14th</div>
 
     <div class="text">August 14th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 76 </p>
+
<ul>
 +
<li>Made ATP standards from assay instructions</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 15th</div>
 
     <div class="text">August 15th</div>
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     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 77 </p>
+
<ul>
 +
<li>Made more LB</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 16th</div>
 
     <div class="text">August 16th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 78 </p>
+
<ul>
 +
<li>Inoculated <i>pgk</i>, <i>pck</i>, DH10B for assay tomorrow</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 17th</div>
 
     <div class="text">August 17th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 79 </p>
+
<ul>
−
    </div>
+
<li>ATP luminescence assay</li>
 +
<li>Inoculated 4 constructs and blank in 𝛥<i>aceE</i> cells for tomorrow</li>
 +
</ul>    </div>
 
     <div class="text">August 18th</div>
 
     <div class="text">August 18th</div>
 
   </div>
 
   </div>
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−
    <p> Stuff that happened on day 80 </p>
+
<ul>
 +
<li>Nothing grew, couldn’t do induction</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 19th</div>
 
     <div class="text">August 19th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 81 </p>
+
<ul>
 +
<li>Inoculated DH10B constructs and control from frozen stock</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 20th</div>
 
     <div class="text">August 20th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 82 </p>
+
<ul>
 +
<li>DH10B electron donor induction, froze pellets, measured OD600</li>
 +
<li>Re-transformed constructs into 𝛥<i>aceE</i> cells</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 21st</div>
 
     <div class="text">August 21st</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 83 </p>
+
<ul>
 +
<li>Sonicated and assayed pellets for biotin (1 minute, 40 amp, 1mL PBS)</li>
 +
<li>Reinoculated 𝛥<i>aceE</i> constructs in all antibiotic combinations to determine problem, in LB and plates</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 22nd</div>
 
     <div class="text">August 22nd</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 84 </p>
+
<ul>
 +
<li>Visited Monsanto</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 23rd</div>
 
     <div class="text">August 23rd</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 85 </p>
+
<ul>
 +
<li>Measured absorbance data from biotin in DH10B cells</li>
 +
<li>Did ATP Assay and got luminescence data</li>
 +
<li>Incubated all four 𝛥<i>aceE</i> electron donor constructs from frozen stock plates and original plates and incubated 𝛥<i>aceE</i> and DH10B</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 24th</div>
 
     <div class="text">August 24th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 86 </p>
+
<ul>
 +
<li>Nothing grew from yesterday's inoculations</li>
 +
<li>Interlab study</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 25th</div>
 
     <div class="text">August 25th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 87 </p>
+
<ul>
 +
<li>Remade competent cells</li>
 +
<li>Finished interlab study (FITC) and sent to iGEM HQ</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">August 26th</div>
 
     <div class="text">August 26th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 95 </p>
+
<ul>
 +
<li>Picked colonies from 4 new 𝛥<i>aceE</i> plates and incubated</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 3rd</div>
 
     <div class="text">September 3rd</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 96 </p>
+
<ul>
 +
<li>𝛥<i>aceE</i> cell induction</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 4th</div>
 
     <div class="text">September 4th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 108 </p>
+
<ul>
 +
<li>Incubated <i>pck</i>, <i>pgk</i>, DH10B from frozen stock</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 16th</div>
 
     <div class="text">September 16th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 109 </p>
+
<ul>
 +
<li>Ran ATP assay</li>
 +
<li>BioBrick digestion and ligation</li>
 +
<li>Sonicated 𝛥<i>aceE</i> pellets</li>
 +
<li>Ran 𝛥<i>aceE</i> cell absorbance scan</li>
 +
<li>Ran biotin assay on 𝛥<i>aceE</i> cells</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 17th</div>
 
     <div class="text">September 17th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 118 </p>
+
<ul>
 +
<li>Transformed ligated <i>petF</i> and <i>pck</i> BioBrick plasmids into DH10B (wrong antibiotic)</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 26th</div>
 
     <div class="text">September 26th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 120 </p>
+
<ul>
 +
<li>Transformed ligated <i>petF</i> and <i>pck</i> BioBrick plasmids into DH10B (kan = right)</li>
 +
<li>Wrapped <i>pck</i> and <i>pgk</i> plasmids to be sent to Cardiff University iGEM</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 28th</div>
 
     <div class="text">September 28th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 121 </p>
+
<ul>
 +
<li>Small colonies grew on all plates</li>
 +
<li>Picked colonies and inoculated</li>
 +
<li>Sent plasmids to Cardiff University iGEM</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 29th</div>
 
     <div class="text">September 29th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 122 </p>
+
<ul>
 +
<li>Miniprep on <i>pck</i> and <i>petF</i> BioBrick</li>
 +
<li>Reusupended TetR-pTet gBlocks</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">September 30th</div>
 
     <div class="text">September 30th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 93 </p>
+
<ul>
 +
<li>Digestion/Ligation/Transformation of pSB1C3 and tetr-ptet BioBrick</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 1st</div>
 
     <div class="text">October 1st</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 94 </p>
+
<ul>
 +
<li>TetR-pTet colonies grew, picked colonies</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 2nd</div>
 
     <div class="text">October 2nd</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 95 </p>
+
<ul>
 +
<li>Miniprepped TetR-pTet BioBrick</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 3rd</div>
 
     <div class="text">October 3rd</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 96 </p>
+
<ul>
 +
<li>digested BioBricks to check bands sizes</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 4th</div>
 
     <div class="text">October 4th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 104 </p>
+
<ul>
 +
<li>Ran PCR on BioBrick plasmids</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 12th</div>
 
     <div class="text">October 12th</div>
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     <div style="height: 200px; width: 400px;">
 
     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 105 </p>
+
<ul>
 +
<li>Ran PCR products on gel</li>
 +
<li>Only <i>petF</i> showed up</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 13th</div>
 
     <div class="text">October 13th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 106 </p>
+
<ul>
 +
<li>digestion of <i>pck</i>, TetR-pTet, and pSB1C3 with EcoRI</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 14th</div>
 
     <div class="text">October 14th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 107 </p>
+
<ul>
 +
<li>Digestion of <i>pck</i>, TetR-pTet, and pSB1C3 with PstI</li>
 +
<li>Ligated <i>pck</i> and TetR-pTet to pSB1C3</li>
 +
<li>Transformed into DH10B</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 15th</div>
 
     <div class="text">October 15th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 108 </p>
+
<ul>
 +
<li>Only TetR-pTet grew</li>
 +
<li>Ran colony PCR with 8 <i>pck</i> colonies and 8 TetR-pTet colonies</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 16th</div>
 
     <div class="text">October 16th</div>
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     <div style="height: 200px; width: 400px;">
 
     </br></br>
 
     </br></br>
−
    <p> Stuff that happened on day 109 </p>
+
<ul>
 +
<li>Ran colony PCR products on a gel but got no bands</li>
 +
<li>Miniprepped pTet1-8</li>
 +
<li>Ran PCR on pTet 1-8 plasmids to check if insert was present</li>
 +
<li>Ran PCR product on gel, got the band!</li>
 +
<li>Prepped <i>petF</i>, <i>pck</i>, and TetR-pTet to be sent to iGEM HQ</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 17th</div>
 
     <div class="text">October 17th</div>
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−
    <p> Stuff that happened on day 110 </p>
+
<ul>
 +
<li>Sent parts to iGEM!</li>
 +
</ul>
 
     </div>
 
     </div>
 
     <div class="text">October 18th</div>
 
     <div class="text">October 18th</div>

Revision as of 17:20, 18 October 2016

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Notebook

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June 4th
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Stuff that happened on day 5

June 5th
6 / 30


June 6th
7 / 30


  • Performed gDNA Extraction on E. Coli MG1655 and Synechocystis 6803
June 7th
8 / 30


  • Designed PCR primers for pgk, pck, fldA, petF, ATP Synthase 1, and ATP Synthase 2
June 8th
9 / 30


Stuff that happened on day 9

June 9th
10 / 30


  • Primers Arrived
  • Ran PCR on ATP Synthase 2 , pgk, fldA, petF, and pdCas9 plasmid
  • Ran all PCR products on gel and recovered ATP Synthase 2, fldA, and petF
  • Ran gradient PCR on pdCas9 plasmid
June 10th
11 / 30


Stuff that happened on day 11

June 11th
12 / 30


Stuff that happened on day 12

June 12th
13 / 30


Made and ran gel for pdCas9

  • Ran PCR on ATP Synthase 1, pgk, and pck
  • Performed gel extraction on yesterday’s ATP Synthase 2
  • Ran pck, pgk, and yesterday’s gradient pdCas9 PCR products on gel and recovered all genes
  • Performed gel extraction on pdCas9, fldA, petF
  • Ran PCR on psc101 and psc102 plasmid
  • Ran psc101 and psc102 PCR products on gel - were recovered
  • Ran gradient PCR on ATP Synthase 1
  • Made agar plates (forgot the antibiotic)
  • Ran golden gate one pot assembly for fldA petF genes and the pdCas9 plasmid
June 13th
14 / 30


  • Ran ATP Synthase 1 PCR products on gel - no bands seen
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Ran PCR on ATP Synthase 1 (3rd times the charm)
  • Ran PCR products on gel - WE SAW BANDS! ATP SYNTHASE 1 RECOVERED
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel Extraction
  • Made new agar plates (with the chloramphenicol)
  • Transformed the petF, fldA plasmids into competent cells
  • Plated colonies on agar plates and set in incubator
June 14th
15 / 30


  • Checked for colonies (both genes were successful)
  • Picked colonies and incubated overnight
  • Ran yesterday’s PCR products on gel - bands were light but all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction
  • Ran PCR products on gel - all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
June 15th
16 / 30


  • Miniprepped plasmids for fldA, petF
  • Ran sequence PCR on both plasmids
  • Ran PCR product on gel to confirm
  • Clean and Concentrated PCR product
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction (for the fourth time)
June 16th
17 / 30


  • Prepared DNA for sequencing
  • Delivered DNA to WashU Med School
  • Added DPnI to psc101 and psc102 gel extract products, incubated for an hour, did Clean and Concentrate, measured conc
  • Ran Golden Gate assembly on pck, pgk, and ATP Synthase
June 17th
18 / 30


  • Electroporation with golden gate products
  • Redid golden gate for pck, pgk, ATP synthase (did not run properly first time)
June 18th
19 / 30


  • Electroporation with better golden gate products
  • Picked colonies for worse golden gate for pck, pgk, ATP synthase
June 19th
20 / 30


  • Miniprep worse ATP, pck, pgk golden gate
  • Seq PCR worse ATP, pck, pgk golden gate
  • Picked colonies for better golden gate for pck, pgk, ATP synthase
June 20th
21 / 30


  • Made frozen stock of better golden gate pck, pgk, ATP synthase
  • Miniprepped better golden gate pck, pgk, ATP synthase (low concentration)
June 21st
22 / 30


  • Ran sequencing PCR for pgk, pck (4 colonies each)
  • Gel Extraction for pgk, pck PCR product (no bands)
June 22nd
23 / 30


  • Ran sequencing PCR for pck and pgk on a gradient
  • Gel Extraction for pck PCR product was successful, not pgk
June 23rd
24 / 30


  • Ran sequencing PCR for pgk on a different gradient
  • Gel Extraction for pgk PCR product was not successful
June 24th
25 / 30


Stuff that happened on day 25

June 25th
26 / 30


  • Picked four new colonies of pgk
  • Started 4 new cultures from frozen stock
June 26th
27 / 30


  • Made frozen stock of the 4 new colonies
  • Miniprepped all 8 pgk cultures and measured concentrations
  • Ran gradient PCR on all 8 pgk plasmids
  • Prepped and sent ATP synthase and pck in for sequencing
June 27th
28 / 30


Stuff that happened on day 28

June 28th
29 / 30


  • Mixed new pgk sequencing primers
June 29th
30 / 30


  • Ran gradient PCR on all 8 pgk plasmids
June 30th
1 / 31


Stuff that happened on day 31

July 1st
2 / 31


Stuff that happened on day 32

July 2nd
3 / 31


Stuff that happened on day 33

July 3rd
4 / 31


4th of July!

July 4th
5 / 31


Stuff that happened on day 35

July 5th
6 / 31


  • Sequencing results for ATP and pck were not good
  • Reran seq PCR on ATP Synthase (bad results)
  • Picked new ATP colonies
July 6th
7 / 31


  • Miniprepped overnight cultures of ATP 5-8 and forgot to make frozen stock
  • Ran PCR on ATP plasmids 5-8
  • PCR for pgk with new primers with gradient
  • Ran pgk PCR product on gels
  • Ran golden gate on pgk PCR gel extract
July 7th
8 / 31


  • Electroporation with golden gate pgk product
July 8th
9 / 31


  • Picked 4 pgk products and inoculated them overnight
July 9th
10 / 31


  • Made frozen stock of new 4 pgk cultures
  • Miniprepped cultures
  • Seq PCR on new cultures
  • All 4 cultures showed correct bands
  • Clean and Concentrate pgk PCR product
  • Prepped pgk for sequencing
July 10th
11 / 31


  • Delivered pgk for sequencing
July 11th
12 / 31


  • New ATP synthase sequencing primers and pck regular primers came in
  • Seq PCR of ATP synthase plasmids with new sequencing primers. No bands
  • Picked 4 new ATP synthase colonies (again)
  • Inoculated MG1655 from frozens stock to make more genomic DNA
July 12th
13 / 31


  • Extracted genomic DNA from MG1655
  • Miniprepped new ATP cultures
  • Ran sequencing PCR on new ATP and got no bands at all. Will use newly transformed cells tomorrow
  • Ran PCR with pck primers, got light bands
  • Reran PCR with pck products
July 13th
14 / 31


  • Ran pck on gel
  • Gel extracted pck and combined gel extracts to improve concentration
  • Golden gate ligation on pck
  • Ran ATP sequencing PCR again with different backbone primers
July 14th
15 / 31


  • Ran gel from yesterday’s ATP PCR. No bands
  • Ran PCR for pfo
  • Incubated pck and BioBrick cells
July 15th
16 / 31


  • Miniprepped pck and biobrock genes (HSP)
July 16th
17 / 31


Stuff that happened on day 47

July 17th
18 / 31


  • Ran ATP seq PCR gradient again. No bands. Done with ATP
  • BioBrick digestion, ligation, transformation with HSP and GFP
  • Ran PCR seq for pck and gel extract
July 18th
19 / 31


  • Delivered pck sequence to med school
  • Ligated backbone for BioBrick
  • Extracted more genomic DNA from MG1655
  • Gradient PCR on pfo
  • Received 𝛥aceE cells from Yale
  • Rehydrated and incubated 𝛥aceE cells
July 19th
20 / 31


  • Ran PCR and gel extract for fldA backbone and petF backbone
  • DPn1 the two backbones
  • golden gate ligation on pfo gene and two backbones
  • Streaked colony from 𝛥aceE plate onto LB/kan plate
July 20th
21 / 31


  • Making media for competent cell protocol
  • Made new LB with MgSO4 for chemical transformation
July 21st
22 / 31


  • Digested BioBrick miniprep to check length
July 22nd
23 / 31


  • Chemically competent cell protocol with 𝛥aceE
  • Miniprepped pfo-petF and fldA-petF
  • Seq PCR miniprepped pfo-petF and fldA=petF
  • Remade LB with MgSO4 with correct concentration
July 23rd
24 / 31


Stuff that happened on day 54

July 24th
25 / 31


  • Restarted comp cell protocol (3rd times the charm)
  • Clean and Concentrate junction 2 for pfo-fldA
  • Conducted HSP test in LB
July 25th
26 / 31


  • Redid HSP test in minimal media M9
  • Recultured BioBrick and DH10B control
July 26th
27 / 31


  • Reset up BioBrick experiment but grew diluted cells with LB instead of M9, so we have to do it again
July 27th
28 / 31


  • Redid HSP test in minimal media M9
July 28th
29 / 31


Stuff that happened on day 59

July 29th
30 / 31


Stuff that happened on day 60

July 30th
31 / 31


Stuff that happened on day 61

July 31st
1 / 31


  • Chemical transformation of pfo-petF, pfo-fldA, petF, fldA into knockout strain, plated 80 and 240 ul, also control
  • Part 1 of interlab study (LUDOX)
  • Transforming 5 interlab plasmids
August 1st
2 / 31


  • Picked and incubated electron donor colonies plated yesterday
  • Picked and incubated interlab colonies plated yesterday (2 per construct)
August 2nd
3 / 31


  • Made frozen stock of incubated colonies and interlab parts
  • Redid BioBrick experiment at 37C

August 3rd
4 / 31


Stuff that happened on day 65

August 4th
5 / 31


Stuff that happened on day 66

August 5th
6 / 31


Stuff that happened on day 67

August 6th
7 / 31


  • Started induction but had to stop bc no frozen stock of fldA-pfo or petF-fldA in DH10B
  • Transformed fldA-pfo and petF-pfo into DH10B
August 7th
8 / 31


  • Made frozen stock of 𝛥aceE
  • Made 60% ethanol
  • Incubated 8 constructs for biotin assay w/ methanol extraction (4 constructs and blank in DH10B and 𝛥aceE)
August 8th
9 / 31


  • Methanol extraction assay (grew cells and froze pellets)
August 9th
10 / 31


  • Assayed petF-aceE cells. Absorbance was too low to be meaningful
August 10th
11 / 31


  • Ran preliminary sonication test
  • Incubated large volume of DH10B
August 11th
12 / 31


  • Ran large sonication test on DH10B to decide protocol
August 12th
13 / 31


  • Incubated large volume of DH10B
August 13th
14 / 31


  • More sonication tests
August 14th
15 / 31


  • Made ATP standards from assay instructions
August 15th
16 / 31


  • Made more LB
August 16th
17 / 31


  • Inoculated pgk, pck, DH10B for assay tomorrow
August 17th
18 / 31


  • ATP luminescence assay
  • Inoculated 4 constructs and blank in 𝛥aceE cells for tomorrow
August 18th
19 / 31


  • Nothing grew, couldn’t do induction
August 19th
20 / 31


  • Inoculated DH10B constructs and control from frozen stock
August 20th
21 / 31


  • DH10B electron donor induction, froze pellets, measured OD600
  • Re-transformed constructs into 𝛥aceE cells
August 21st
22 / 31


  • Sonicated and assayed pellets for biotin (1 minute, 40 amp, 1mL PBS)
  • Reinoculated 𝛥aceE constructs in all antibiotic combinations to determine problem, in LB and plates
August 22nd
23 / 31


  • Visited Monsanto
August 23rd
24 / 31


  • Measured absorbance data from biotin in DH10B cells
  • Did ATP Assay and got luminescence data
  • Incubated all four 𝛥aceE electron donor constructs from frozen stock plates and original plates and incubated 𝛥aceE and DH10B
August 24th
25 / 31


  • Nothing grew from yesterday's inoculations
  • Interlab study
August 25th
26 / 31


  • Remade competent cells
  • Finished interlab study (FITC) and sent to iGEM HQ
August 26th
27 / 31


Stuff that happened on day 88

August 27th
28 / 31


Stuff that happened on day 89

August 28th
29 / 31


Stuff that happened on day 90

August 29th
30 / 31


Stuff that happened on day 91

August 30th
31 / 31


Stuff that happened on day 92

August 31st
1 / 30


Stuff that happened on day 93

September 1st
2 / 30


Stuff that happened on day 94

September 2nd
3 / 30


  • Picked colonies from 4 new 𝛥aceE plates and incubated
September 3rd
4 / 30


  • 𝛥aceE cell induction
September 4th
5 / 30


Stuff that happened on day 97

September 5th
6 / 30


Stuff that happened on day 98

September 6th
7 / 30


Stuff that happened on day 99

September 7th
8 / 30


Stuff that happened on day 100

September 8th
9 / 30


Stuff that happened on day 101

September 9th
10 / 30


Stuff that happened on day 102

September 10th
11 / 30


Stuff that happened on day 103

September 11th
12 / 30


Stuff that happened on day 104

September 12th
13 / 30


Stuff that happened on day 105

September 13th
14 / 30


Stuff that happened on day 106

September 14th
15 / 30


Stuff that happened on day 107

September 15th
16 / 30


  • Incubated pck, pgk, DH10B from frozen stock
September 16th
17 / 30


  • Ran ATP assay
  • BioBrick digestion and ligation
  • Sonicated 𝛥aceE pellets
  • Ran 𝛥aceE cell absorbance scan
  • Ran biotin assay on 𝛥aceE cells
September 17th
18 / 30


Stuff that happened on day 110

September 18th
19 / 30


Stuff that happened on day 111

September 19th
20 / 30


Stuff that happened on day 112

September 20th
21 / 30


Stuff that happened on day 113

September 21st
22 / 30


Stuff that happened on day 114

September 22nd
23 / 30


Stuff that happened on day 115

September 23rd
24 / 30


Stuff that happened on day 116

September 24th
25 / 30


Stuff that happened on day 117

September 25th
26 / 30


  • Transformed ligated petF and pck BioBrick plasmids into DH10B (wrong antibiotic)
September 26th
27 / 30


Stuff that happened on day 119

September 27th
28 / 30


  • Transformed ligated petF and pck BioBrick plasmids into DH10B (kan = right)
  • Wrapped pck and pgk plasmids to be sent to Cardiff University iGEM
September 28th
29 / 30


  • Small colonies grew on all plates
  • Picked colonies and inoculated
  • Sent plasmids to Cardiff University iGEM
September 29th
30 / 30


  • Miniprep on pck and petF BioBrick
  • Reusupended TetR-pTet gBlocks
September 30th
1 / 31


  • Digestion/Ligation/Transformation of pSB1C3 and tetr-ptet BioBrick
October 1st
2 / 31


  • TetR-pTet colonies grew, picked colonies
October 2nd
3 / 31


  • Miniprepped TetR-pTet BioBrick
October 3rd
4 / 31


  • digested BioBricks to check bands sizes
October 4th
5 / 31


Stuff that happened on day 97

October 5th
6 / 31


Stuff that happened on day 98

October 6th
7 / 31


Stuff that happened on day 99

October 7th
8 / 31


Stuff that happened on day 100

October 8th
9 / 31


Stuff that happened on day 101

October 9th
10 / 31


Stuff that happened on day 102

October 10th
11 / 31


Stuff that happened on day 103

October 11th
12 / 31


  • Ran PCR on BioBrick plasmids
October 12th
13 / 31


  • Ran PCR products on gel
  • Only petF showed up
October 13th
14 / 31


  • digestion of pck, TetR-pTet, and pSB1C3 with EcoRI
October 14th
15 / 31


  • Digestion of pck, TetR-pTet, and pSB1C3 with PstI
  • Ligated pck and TetR-pTet to pSB1C3
  • Transformed into DH10B
October 15th
16 / 31


  • Only TetR-pTet grew
  • Ran colony PCR with 8 pck colonies and 8 TetR-pTet colonies
October 16th
17 / 31


  • Ran colony PCR products on a gel but got no bands
  • Miniprepped pTet1-8
  • Ran PCR on pTet 1-8 plasmids to check if insert was present
  • Ran PCR product on gel, got the band!
  • Prepped petF, pck, and TetR-pTet to be sent to iGEM HQ
October 17th
18 / 31


  • Sent parts to iGEM!
October 18th
19 / 31


Stuff that happened on day 111

October 19th
20 / 31


Stuff that happened on day 112

October 20th
21 / 31


Stuff that happened on day 113

October 21st
22 / 31


Stuff that happened on day 114

October 22nd
23 / 31


Stuff that happened on day 115

October 23rd
24 / 31


Stuff that happened on day 116

October 24th
25 / 31


Stuff that happened on day 117

October 25th
26 / 31


Stuff that happened on day 118

October 26th
27 / 31


Stuff that happened on day 119

October 27th
28 / 31


Stuff that happened on day 120

October 28th
29 / 31


Stuff that happened on day 121

October 29th
30 / 31


Stuff that happened on day 122

October 30th
31 / 31


Stuff that happened on day 122

October 31st