Difference between revisions of "Team:Northwestern/09 01"

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   <article>
 
   <article>
 
     <h1>Thursday, September 1<sup>st</sup></h3>
 
     <h1>Thursday, September 1<sup>st</sup></h3>
−
     <h2>Agenda:</h2>
+
<h2>Transformation Results:</h2>
−
 
+
    <p>The gRNA constructs are assembling; Cas9 + YcdO once again failed to assemble.</p>
 +
    <div class="row">
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/b/bf/T--Northwestern--09_01_2.png" width="460" height="464" alt=""/>
 +
        <p><strong>Figure 1:</strong> mRFP in pSB1T3 transformation</p>
 +
      </div>
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/6/63/T--Northwestern--09_01_3.png" width="478" height="469" alt=""/>
 +
        <p><strong>Figure 2:</strong> mRFP in pSB1T3 transformation 2</p>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/0/01/T--Northwestern--09_01_4.png" width="1601" height="1615" alt=""/>
 +
        <p><strong>Figure 3:</strong> Gibson negative control (no insert)</p>
 +
      </div>
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/3/36/T--Northwestern--09_01_5.png" width="1587" height="1557" alt=""/>
 +
        <p><strong>Figure 4:</strong> Gibson mix positive control</p>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/2/25/T--Northwestern--09_01_6.png" width="1558" height="1600" alt=""/>
 +
        <p><strong>Figure 5:</strong> gRNA Gibson retransformation</p>
 +
      </div>
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/c/cd/T--Northwestern--09_01_7.png" width="1600" height="1589" alt=""/>
 +
        <p><strong>Figure 6:</strong> Transformation positive control</p>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-6"><img src="https://static.igem.org/mediawiki/2016/6/6c/T--Northwestern--09_01_8.png" width="1605" height="1648" alt=""/>
 +
        <p><strong>Figure 7:</strong> YcdO + Cas9 Gibson</p>
 +
      </div>
 +
</div>
 +
     <h2>Tasks:</h2>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Jordan</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Imaged periplasm fractions and ClyA-GFP with EVOS
 +
            <div class="row">
 +
              <div class="col-xs-6"><img src="https://static.igem.org/mediawiki/2016/c/cf/T--Northwestern--09_01_1.png" width="1360" height="1024" alt=""/>
 +
                <p><strong>Figure 1:</strong> Whole cell ClyA-GFP</p>
 +
              </div>
 +
            </div>
 +
          </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Michelle</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Transferred TetR-Cas9 induced cultures to 10 mL tubes</li>
 +
          <li>Miniprepped GFP 1–3 in storage vectors </li>
 +
          <ul>
 +
            <li>GFP1 colony A: 77.7 ng/uL, 260/280: 1.90, 260/230: 2.18</li>
 +
            <li>GFP1 colony B: 223.0 ng/uL, 260/280: 1.76, 260/230: 1.25</li>
 +
            <li>GFP2 colony A: 65.2 nh/uL, 260/280: 1.92, 260/230: 2.32</li>
 +
            <li>GFP2 colony B: 132.1 ng/uL, 260/280: 1.89, 260/230: 2.26</li>
 +
            <li>GFP3 colony A: 19.3 ng/uL, 260/280: 1.90, 260/230: 1.50</li>
 +
            <li>GFP3 colony B: 19.0 ng/uL, 260/280: 1.76, 260/230: 0.71</li>
 +
          </ul>
 +
          <li>Grew 5mL overnight culture (x3) of gRNA from retransformed plate 5 mL LB + 5uL 1000X Tet</li>
 +
          <ul>
 +
            <li>Miniprep tomorrow for electroporation cotransformation next week</li>
 +
          </ul>
 +
          <li>Grew 5mL overnight culture (x2) of Cas9 from glycerol stock 5 mL LB + 5uL 1000X Cam</li>
 +
          <ul>
 +
            <li>Miniprep tomorrow for electroporation cotransformation next week </li>
 +
          </ul>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Sam</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li>Prepped questions for Dr. Scheetz</li>
 +
          <li>Looked into Australia protocol</li>
 +
          <li>Interviewed Dr. Scheetz </li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
    <div class="row">
 +
      <div class="col-sm-2">
 +
        <p>Tasfia</p>
 +
      </div>
 +
      <div class="col-sm-10">
 +
        <ul>
 +
          <li> Made sequencing rxn mixtures for (~500 ng) gRNA-mRFP Gibson product based on Paul’s notes from 7.26.16 using the following primers</li>
 +
          <ul><li>VR (Biobrick Rev)</li>
 +
          <li>Cas9-Seq4 (Fwd)</li>
 +
          <li>VF (Biobrick Fwd)</li></ul>
 +
          <li>Gibson Reaction for Ycdo into “Cas9-Lrz-SS 1:3” linearized on 8.29.16</li>
 +
          <li>New conditions for Gibson:</li>
 +
          <ul><li>Ran 5:1 molar ratio of insert-to-backbone, 15 minutes at 50&#176;C in thermal cycler</li>
 +
          <li>“Cas9-YcdO”</li>
 +
          <ul><li>1.34 µL backbone</li>
 +
          <li>0.14 µL insert (Ycdo)</li>
 +
          <li>3.52 µL water</li>
 +
          <li>5 µL Gibson mix</li></ul>
 +
          <li>Negative Control</li>
 +
          <ul><li>1.34 µL backbone</li>
 +
          <li>3.66 µL water</li>
 +
          <li>5 µL Gibson mix</li></ul>
 +
          <li>Positive Control</li>
 +
          <ul><li>5µL (+) control</li>
 +
          <li>5µL master mix</li></ul></ul>
 +
          <li>Transformation</li>
 +
        <ul> <li>1 µL (+) transformation control (CamR)</li>
 +
          <li>2 µL Gibson (+) control (AmpR)</li>
 +
          <li>2 µL Gibson (-) Control (CamR)</li>
 +
          <li>3 µL Gibson Product (CamR)</li></ul>
 +
         
 +
          <li>Worked on presentation for 9.2.16</li>
 +
          <li>Attempted to take inventory of Gibson products (check -20&#176;C inventory spreadsheet)</li>
 +
        </ul>
 +
      </div>
 +
    </div>
 +
</article>
 
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Revision as of 01:55, 16 October 2016

Notebook

Thursday, September 1st

Transformation Results:

The gRNA constructs are assembling; Cas9 + YcdO once again failed to assemble.

Figure 1: mRFP in pSB1T3 transformation

Figure 2: mRFP in pSB1T3 transformation 2

Figure 3: Gibson negative control (no insert)

Figure 4: Gibson mix positive control

Figure 5: gRNA Gibson retransformation

Figure 6: Transformation positive control

Figure 7: YcdO + Cas9 Gibson

Tasks:

Jordan

  • Imaged periplasm fractions and ClyA-GFP with EVOS

    Figure 1: Whole cell ClyA-GFP

Michelle

  • Transferred TetR-Cas9 induced cultures to 10 mL tubes
  • Miniprepped GFP 1–3 in storage vectors
    • GFP1 colony A: 77.7 ng/uL, 260/280: 1.90, 260/230: 2.18
    • GFP1 colony B: 223.0 ng/uL, 260/280: 1.76, 260/230: 1.25
    • GFP2 colony A: 65.2 nh/uL, 260/280: 1.92, 260/230: 2.32
    • GFP2 colony B: 132.1 ng/uL, 260/280: 1.89, 260/230: 2.26
    • GFP3 colony A: 19.3 ng/uL, 260/280: 1.90, 260/230: 1.50
    • GFP3 colony B: 19.0 ng/uL, 260/280: 1.76, 260/230: 0.71
  • Grew 5mL overnight culture (x3) of gRNA from retransformed plate 5 mL LB + 5uL 1000X Tet
    • Miniprep tomorrow for electroporation cotransformation next week
  • Grew 5mL overnight culture (x2) of Cas9 from glycerol stock 5 mL LB + 5uL 1000X Cam
    • Miniprep tomorrow for electroporation cotransformation next week

Sam

  • Prepped questions for Dr. Scheetz
  • Looked into Australia protocol
  • Interviewed Dr. Scheetz

Tasfia

  • Made sequencing rxn mixtures for (~500 ng) gRNA-mRFP Gibson product based on Paul’s notes from 7.26.16 using the following primers
    • VR (Biobrick Rev)
    • Cas9-Seq4 (Fwd)
    • VF (Biobrick Fwd)
  • Gibson Reaction for Ycdo into “Cas9-Lrz-SS 1:3” linearized on 8.29.16
  • New conditions for Gibson:
    • Ran 5:1 molar ratio of insert-to-backbone, 15 minutes at 50°C in thermal cycler
    • “Cas9-YcdO”
      • 1.34 µL backbone
      • 0.14 µL insert (Ycdo)
      • 3.52 µL water
      • 5 µL Gibson mix
    • Negative Control
      • 1.34 µL backbone
      • 3.66 µL water
      • 5 µL Gibson mix
    • Positive Control
      • 5µL (+) control
      • 5µL master mix
  • Transformation
    • 1 µL (+) transformation control (CamR)
    • 2 µL Gibson (+) control (AmpR)
    • 2 µL Gibson (-) Control (CamR)
    • 3 µL Gibson Product (CamR)
  • Worked on presentation for 9.2.16
  • Attempted to take inventory of Gibson products (check -20°C inventory spreadsheet)