Difference between revisions of "Team:Tufts/Results"

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<html>
 
<html>
  
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<h2> Project Results</h2>
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<p>The first few steps of the project involved purifying and checking both of the genes for Cas9 and aTcdB.  This involved purifying the products of PCR and running them on agarose gels, as well as using a spectrophotometer to identify the concentration and purity of DNA recovered.</p>
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&nbsp;&nbsp;1. pHis1522-aTcdB<br>
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<table>
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<tr>
 +
<td>Sample</td>
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<td>Concentration (ng/μL)</td>
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</tr>
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<tr>
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<td>A</td>
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<td>135.5</td>
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</tr>
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<tr>
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<td>B</td>
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<td>170.2</td>
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</tr>
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</table>
  
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<p>Here you can describe the results of your project and your future plans. </p>
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    2. pHsp70-Cas9
  
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<h5>What should this page contain?</h5>
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<table>
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<ul>
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<tr>
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<li> Clearly and objectively describe the results of your work.</li>
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<td>Sample</td>
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<li> Future plans for the project </li>
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<td>Concentration (ng/μL)</td>
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<li> Considerations for replicating the experiments </li>
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</tr>
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</ul>
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<tr>
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<td>A</td>
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<td>32.9</td>
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</tr>
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<tr>
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<td>B</td>
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<td>33.3</td>
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</tr>
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<tr>
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<td>C</td>
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<td>118.3</td>
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</tr>
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</table>
  
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<center><img src = "https://static.igem.org/mediawiki/2015/3/3e/Tuftsresultsgel1.png"  height="30%" width="30%"/></center>
  
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The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples.
  
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The genes were then ligated into vectors and transformed into JM109 E. Coli cells in order to replicate the vectors.  The bacteria were left to grow overnight, and then the plasmid was isolated from them.
  
  
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<h4> Project Achievements </h4>
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pHsp70-Cas9
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<table>
 +
<tr>
 +
<td>Sample</td>
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<td>Concentration (ng/μL)</td>
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</tr>
 +
<tr>
 +
<td>A</td>
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<td>45.7</td>
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</tr>
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<tr>
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<td>B</td>
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<td>46.3</td>
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</tr>
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<tr>
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<td>C</td>
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<td>40.4</td>
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</tr>
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</table>
  
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<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
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Finally, the NLS-tagged Cas9 PCR product was combined with the linearized pHis1522-aTcdB vector in a Gibson assembly reaction. This reaction was transformed into NEB 5-alpha E. coli. Several transformants were used to inoculate liquid cultures, whose plasmids were subsequently isolated.
  
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<ul>
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Concentrations of pHis1522-Cas9-aTcdB Minipreps
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<li>A list of linked bullet points of the successful results during your project</li>
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<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
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<table>
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</ul>
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<tr>
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<td>Sample</td>
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<td>Concentration (ng/μL)</td>
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</tr>
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<tr>
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<td>1</td>
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<td>49.9</td>
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</tr>
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<tr>
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<td>2</td>
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<td>72.1</td>
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</tr>
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<tr>
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<td>3</td>
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<td>1702.6</td>
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</tr>
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<tr>
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<td>4</td>
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<td>426.6</td>
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</tr>
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<tr>
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<td>5</td>
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<td>42.7</td>
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</tr>
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</table>
 +
 
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A PCR to amplify the Cas9 portion was performed to confirm sucessful assemly. Below is the gel of samples 1-5. A faint band (not clearly visible in image) at ~4.2 kb in sample 3 indicates Cas9's presence.
 +
 
 +
<center><img src = "https://static.igem.org/mediawiki/2015/8/86/Tuftsresultsgel2.png" height="30%" width="30%"/></center>
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 +
 
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In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA for T7 driven transcription.  This construct was obtained as a gBlock, amplified, and inserted into the pSB1C3 vector with restriction cloning using EcoRI and PstI.  
 +
<center><img src = "https://static.igem.org/mediawiki/2015/f/f1/Tuftsplate.jpg" height="30%" width="30%"/></center>
 +
 
 +
Colonies lacking RFP were used to inoculate liquid cultures, whose plasmids were ultimately purified and sequenced. Sequencing confirmed successful insertion. This construct now constitutes our Biobrick part BBa_K1818000.
 +
 
 +
Unfortunately, an attempt to insert a pair of phosphorylated annealed oligos into this template was unsuccessful, as confirmed by sequencing which shows that the BbsI did not cut the vector or that the vector re-ligated.
  
  
  
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<h4>Inspiration</h4>
 
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<p>See how other teams presented their results.</p>
 
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<ul>
 
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<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
 
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<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
 
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</ul>
 
  
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</div>
 
 
</html>
 
</html>

Latest revision as of 03:32, 19 September 2015

The first few steps of the project involved purifying and checking both of the genes for Cas9 and aTcdB. This involved purifying the products of PCR and running them on agarose gels, as well as using a spectrophotometer to identify the concentration and purity of DNA recovered.

  1. pHis1522-aTcdB
Sample Concentration (ng/μL)
A 135.5
B 170.2
2. pHsp70-Cas9
Sample Concentration (ng/μL)
A 32.9
B 33.3
C 118.3
The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples. The genes were then ligated into vectors and transformed into JM109 E. Coli cells in order to replicate the vectors. The bacteria were left to grow overnight, and then the plasmid was isolated from them. pHsp70-Cas9
Sample Concentration (ng/μL)
A 45.7
B 46.3
C 40.4
Finally, the NLS-tagged Cas9 PCR product was combined with the linearized pHis1522-aTcdB vector in a Gibson assembly reaction. This reaction was transformed into NEB 5-alpha E. coli. Several transformants were used to inoculate liquid cultures, whose plasmids were subsequently isolated. Concentrations of pHis1522-Cas9-aTcdB Minipreps
Sample Concentration (ng/μL)
1 49.9
2 72.1
3 1702.6
4 426.6
5 42.7
A PCR to amplify the Cas9 portion was performed to confirm sucessful assemly. Below is the gel of samples 1-5. A faint band (not clearly visible in image) at ~4.2 kb in sample 3 indicates Cas9's presence.
In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA for T7 driven transcription. This construct was obtained as a gBlock, amplified, and inserted into the pSB1C3 vector with restriction cloning using EcoRI and PstI.
Colonies lacking RFP were used to inoculate liquid cultures, whose plasmids were ultimately purified and sequenced. Sequencing confirmed successful insertion. This construct now constitutes our Biobrick part BBa_K1818000. Unfortunately, an attempt to insert a pair of phosphorylated annealed oligos into this template was unsuccessful, as confirmed by sequencing which shows that the BbsI did not cut the vector or that the vector re-ligated.