Team:Concordia/Notebook

From 2013.igem.org

(Difference between revisions)
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     <li><a href="https://2013.igem.org/Team:Concordia">Home</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia">Home</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Project">Project</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Project">Project</a></li>
 +
      <ul class="project-list">
 +
          <li><a href="https://2013.igem.org/Team:Concordia/GasClock">Gas Clock</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Logic">Logic</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Interface">Interface</a></li>
 +
          <li><a href="https://2013.igem.org/Team:Concordia/Memory">Memory</a></li>
 +
      </ul>
     <li><a href="https://2013.igem.org/Team:Concordia/Team">Team</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Team">Team</a></li>
     <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Concordia">Official Profile</a></li>
     <li><a href="https://igem.org/Team.cgi?year=2013&team_name=Concordia">Official Profile</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Parts">Registry</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Parts">Registry</a></li>
 +
    <li><a href="https://2013.igem.org/Team:Concordia/HumanPractices">Human Practices</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Modeling">Modeling</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Modeling">Modeling</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Notebook">Notebook</a></li>
     <li><a href="https://2013.igem.org/Team:Concordia/Notebook">Notebook</a></li>
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   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Failure for PCR amplification of Ethylene Cassette synthesis genes.  </h2>
+
       <h2 class="log-title">Gene Regulatory Network Completed</h2>
-
       <h4 class="log-time">Sept, 13th</h4>
+
       <h4 class="log-time">June, 27th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/c9/Sept_13.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/74/June_27.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Yeast cell strain, for yeast assembly of the ethylene cassette genes, was made and thus needed its parts to assemble. The amplification of ethylene cassette genes had been previously attempted and failed. This same PCR was then attempted again on a different PCR machine and using a temperature gradient from 52 degrees to 60 degrees Celsius; the results was still unsuccessful. Upon review all reverse primers used for said genes were incorrect; this is one definite source of error!</p>
+
       <p class="log-long-desc">After 2 months of meetings, discussion and research we have finalized our genetic regulatory network. Next stop… wet lab!</p>
     </div>
     </div>
   </div>
   </div>
Line 85: Line 92:
   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">GFP amplified in mini-prep samples</h2>
+
       <h2 class="log-title">Amplification of ETR1 and EnvZ Fragments Successful</h2>
-
       <h4 class="log-time">Sept, 12th</h4>
+
       <h4 class="log-time">July, 26th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7c/Sept_12.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/3/32/July_26.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.</p>
+
       <p class="log-long-desc">We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.</p>
     </div>
     </div>
   </div>
   </div>
Line 100: Line 107:
   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Mini-Prep of PompC transformed E.coli cells. </h2>
+
       <h2 class="log-title">Generated Fusions</h2>
-
      <h4 class="log-time">Sept, 10th</h4>
+
       <h4 class="log-time">July, 29th</h4>
-
    </div>
+
-
   
+
-
    <div class="log-desc">
+
-
      <p class="log-long-desc">Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures. </p>
+
-
    </div>
+
-
  </div>
+
-
 
+
-
  <div class="log col-md-9 log-left">
+
-
    <div class="log-title-wrap">
+
-
      <h2 class="log-title">Overlapping PCR of RBS/Plac and Fusions was unsuccessful</h2>
+
-
       <h4 class="log-time">Sept, 9th</h4>
+
-
    </div>
+
-
   
+
-
    <div class="log-img-wrap">
+
-
      <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7d/Sept_9_1.PNG"/>
+
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/1/17/Sept_9_2.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/0/01/July_29.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.</p>
+
       <p class="log-long-desc">We successfully extracted the amplicons (from 07-26) from 1% Agarose gel to be used in subsequent overlapping PCR experiments. This was a fundamental step as any residual primers within the sample will make it difficult to perform the overlapping PCR.</p>
-
    </div>
+
-
  </div>
+
-
 
+
-
  <div class="log col-md-9 log-right">
+
-
    <div class="log-title-wrap">
+
-
      <h2 class="log-title">Overlapping PCR of RBS/Plac and Fusions was unsuccessful</h2>
+
-
      <h4 class="log-time">Sept, 10th</h4>
+
-
    </div>
+
-
   
+
-
    <div class="log-desc">
+
-
      <p class="log-long-desc">Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.</p>
+
     </div>
     </div>
   </div>
   </div>
Line 141: Line 122:
   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.</h2>
+
       <h2 class="log-title">Generated Fusions</h2>
-
       <h4 class="log-time">Sept, 3rd</h4>
+
       <h4 class="log-time">Aug, 1st</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/2/28/Sept_3.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/cd/Aug_1.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.</p>
+
       <p class="log-long-desc">Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!</p>
     </div>
     </div>
   </div>
   </div>
Line 156: Line 137:
   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Ethanol Purification of ETR1, PLac, and PompC-GFP</h2>
+
       <h2 class="log-title">Amplified EnvZ, but No LIT fusion</h2>
-
       <h4 class="log-time">Aug, 22nd</h4>
+
       <h4 class="log-time">Aug, 6th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/4/47/Aug_22.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/a/ac/Aug_6.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">ETR1, PLac and PompC-GFP were Ethanol Purified.</p>
+
       <p class="log-long-desc">We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.</p>
     </div>
     </div>
   </div>
   </div>
Line 171: Line 152:
   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP. </h2>
+
       <h2 class="log-title">Ethanol purification of EnvZ, and Fusions DOM/CON</h2>
-
       <h4 class="log-time">Aug, 21st</h4>
+
       <h4 class="log-time">Aug, 7th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/5/51/Aug_21.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/e/ec/Aug_7.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!</p>
+
       <p class="log-long-desc">Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.</p>
     </div>
     </div>
   </div>
   </div>
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   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Phire Discontinued</h2>
+
       <h2 class="log-title">Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP. </h2>
-
       <h4 class="log-time">Aug, 12th</h4>
+
       <h4 class="log-time">Aug, 21st</h4>
 +
    </div>
 +
   
 +
    <div class="log-img-wrap">
 +
      <img class="log-img" src="https://static.igem.org/mediawiki/2013/5/51/Aug_21.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">The Phire DNA polymerase sample we used was obtained from the Dr.Martin lab. Since it was only a trial sample, more Phire DNA polymerase needed to be ordered. We absolutely wanted to order Phire DNA polymerase since it had proven to be successful previously (Aug 01). But when we attempted to order it we figured out that the original Phire, and its second generation 'Phire Hot start Green polymerase' had been discontinued.</p>
+
       <p class="log-long-desc">Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!</p>
     </div>
     </div>
   </div>
   </div>
Line 212: Line 197:
   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Ethanol purification of EnvZ, and Fusions DOM/CON</h2>
+
       <h2 class="log-title">Ethanol Purification of ETR1, PLac, and PompC-GFP</h2>
-
       <h4 class="log-time">Aug, 7th</h4>
+
       <h4 class="log-time">Aug, 22nd</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/e/ec/Aug_7.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/4/47/Aug_22.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.</p>
+
       <p class="log-long-desc">ETR1, PLac and PompC-GFP were Ethanol Purified.</p>
     </div>
     </div>
   </div>
   </div>
Line 227: Line 212:
   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Amplified EnvZ, but No LIT fusion</h2>
+
       <h2 class="log-title">Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.</h2>
-
       <h4 class="log-time">Aug, 6th</h4>
+
       <h4 class="log-time">Sept, 3rd</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/a/ac/Aug_6.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/2/28/Sept_3.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.</p>
+
       <p class="log-long-desc">Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.</p>
     </div>
     </div>
   </div>
   </div>
Line 242: Line 227:
   <div class="log col-md-9 log-right">
   <div class="log col-md-9 log-right">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Low Purification Yield; Fusions</h2>
+
       <h2 class="log-title">Mini-Prep of PompC transformed E.coli cells. </h2>
-
       <h4 class="log-time">Aug, 2nd</h4>
+
       <h4 class="log-time">Sept, 10th</h4>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Our Fusions that were generated using 'Phire DNA polymerase' were Gel Purified and visualized using Gel Electrophoresis. What we saw was not good news, Fusions LIT and CON were not concentrated enough. DOM, and CON were usable but LIT was not. LIT fusion has to be amplified.</p>
+
       <p class="log-long-desc">Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures. </p>
     </div>
     </div>
   </div>
   </div>
Line 253: Line 238:
   <div class="log col-md-9 log-left">
   <div class="log col-md-9 log-left">
     <div class="log-title-wrap">
     <div class="log-title-wrap">
-
       <h2 class="log-title">Generated Fusions</h2>
+
       <h2 class="log-title">GFP amplified in mini-prep samples</h2>
-
       <h4 class="log-time">Aug, 1st</h4>
+
       <h4 class="log-time">Sept, 12th</h4>
     </div>
     </div>
      
      
     <div class="log-img-wrap">
     <div class="log-img-wrap">
-
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/c/cd/Aug_1.PNG"/>
+
       <img class="log-img" src="https://static.igem.org/mediawiki/2013/7/7c/Sept_12.PNG"/>
     </div>
     </div>
      
      
     <div class="log-desc">
     <div class="log-desc">
-
       <p class="log-long-desc">Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!</p>
+
       <p class="log-long-desc">Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.</p>
     </div>
     </div>
   </div>
   </div>

Revision as of 00:03, 28 September 2013

Notebook

Gene Regulatory Network Completed

June, 27th

After 2 months of meetings, discussion and research we have finalized our genetic regulatory network. Next stop… wet lab!

Amplification of ETR1 and EnvZ Fragments Successful

July, 26th

We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.

Generated Fusions

July, 29th

We successfully extracted the amplicons (from 07-26) from 1% Agarose gel to be used in subsequent overlapping PCR experiments. This was a fundamental step as any residual primers within the sample will make it difficult to perform the overlapping PCR.

Generated Fusions

Aug, 1st

Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!

Amplified EnvZ, but No LIT fusion

Aug, 6th

We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.

Ethanol purification of EnvZ, and Fusions DOM/CON

Aug, 7th

Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.

Amplification/Ethanol Purification of TetR and RhlR. Ethanol Purification of PompC-GFP

Aug, 13th

PCR amplification of interface genes was commenced; RhlR was successfully amplified and purified. RhlR was quantified to be 360 ng/ul. Gas clock component 'TetR' was also successfully amplified, and later purified. TetR quantification yield was 216 ng/ul. Additionally, PompC-GFP was successfully ethanol purified and quantified to be approximately 270 ng/ul.

Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP.

Aug, 21st

Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!

Ethanol Purification of ETR1, PLac, and PompC-GFP

Aug, 22nd

ETR1, PLac and PompC-GFP were Ethanol Purified.

Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.

Sept, 3rd

Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.

Mini-Prep of PompC transformed E.coli cells.

Sept, 10th

Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures.

GFP amplified in mini-prep samples

Sept, 12th

Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.