Difference between revisions of "Team:HokkaidoU Japan/Results"

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<h1>Results</h1>
 
<h1>Results</h1>
 
<p>Until the Wiki freeze, we tried to construct plasmid with the insert we designed and managed to combine plasmid with INSERT 1, 2 or 3(we couldn't insert INSERT 4, which is a control of the INSERT 2 or 3).</p>
 
<p>Until the Wiki freeze, we tried to construct plasmid with the insert we designed and managed to combine plasmid with INSERT 1, 2 or 3(we couldn't insert INSERT 4, which is a control of the INSERT 2 or 3).</p>
−
  <p>To assay our construct, we cultured E. coli with IPTG induction, purified the crude extract by the His-tag and condensed them using </p>
+
  <p>To assay our construct, we cultured E. coli with IPTG induction, purified the crude extract by the His-tag and condensed them.</p>
 
  <p>When the expressed protein (both INSERT 2 and 3) were cut at the signal peptides, the molecular weight becomes 52367.76 or 48575.69 respectively.</p>
 
  <p>When the expressed protein (both INSERT 2 and 3) were cut at the signal peptides, the molecular weight becomes 52367.76 or 48575.69 respectively.</p>
 
  <p>The figure bellow is the result of SDS-PAGE. We applied purified INSERT 2 (left) and INSERT 3 (right, not visible)and found that the band of INSERT 2 is at the correct place.</p>
 
  <p>The figure bellow is the result of SDS-PAGE. We applied purified INSERT 2 (left) and INSERT 3 (right, not visible)and found that the band of INSERT 2 is at the correct place.</p>
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<p>We assumed two possibility that explain why we couldn't get the band of INSERT 3; the concentration of protein is too small, the His-tag didn't interacted with the Nickel column and flowed  through.</p>
 
<p>We assumed two possibility that explain why we couldn't get the band of INSERT 3; the concentration of protein is too small, the His-tag didn't interacted with the Nickel column and flowed  through.</p>
 
<p>To determine the reason why we couldn't see the band, we conducted phytase activity assay with the crude extract, purified and condensed products, and the flow through of purification process.</p>
 
<p>To determine the reason why we couldn't see the band, we conducted phytase activity assay with the crude extract, purified and condensed products, and the flow through of purification process.</p>
−
<p>As the table and figure above shows, we can see high phytase activity in the flow through and concluded that His-tag didn't interacted with the Nickel, probably because the His-tag was not exposed outside. Simultaneously, we can assume that self-Assembling Peptides helped His-tag being exposed and interacting with Nickel.</p>
+
<p>As the table and figure above shows, we can see high phytase activity in the flow through and concluded that His-tag didn't interacted with the Nickel, probably because the His-tag was not exposed outside. Simultaneously, we can assume that self-Assembling Peptides (SAPs) helped His-tag being exposed and interacting with Nickel.</p>
 
<img src="https://static.igem.org/mediawiki/2017/thumb/4/42/T--HokkaidoU_Japan--SDS-PAGE02.JPG/800px-T--HokkaidoU_Japan--SDS-PAGE02.JPG" width="700" height="auto">
 
<img src="https://static.igem.org/mediawiki/2017/thumb/4/42/T--HokkaidoU_Japan--SDS-PAGE02.JPG/800px-T--HokkaidoU_Japan--SDS-PAGE02.JPG" width="700" height="auto">
−
<p>We applied crude extract of INSERT 2 (left) and INSERT 3 (right). </p>
+
<p>We applied crude extract of INSERT 2 (left) and INSERT 3. (right). The difference of mobility between INSERT 2 and 3 is very small but may be slightly different. (Difficult to judge)</p>
−
<p>Here you can describe the results of your project and your future plans. </p>
+
  
−
<h5>What should this page contain?</h5>
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<h5>Phytase thermostability</h5>
 
<ul>
 
<ul>
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<li> Clearly and objectively describe the results of your work.</li>
+
<p> We assayed thermostability of several kind of phytase; </p>
−
<li> Future plans for the project. </li>
+
<p>Though the degree of deactivation is different according to kind of phytase, the characteristic that remaining activity after 1 hour heat shock drops is common.</p>
−
<li> Considerations for replicating the experiments. </li>
+
<p>From the comparison between INSERT 2 and 3, we can see that activity of phytase from insert 3 drops more drastically than that of from INSERT 2, suggesting that SAPs will improve thermostability of phytase.</p>
 
</ul>
 
</ul>
−
 
−
<h5>You should also describe what your results mean: </h5>
 
−
 
−
<ul>
 
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<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
 
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<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
 
−
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
 
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</ul>
 
−
 
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</div>
 
−
 
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<div class="clear"></div>
 
−
 
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<div class="column half_size" >
 
  
  
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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>
 
<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>
−
 
 
<ul>
 
<ul>
−
<li>A list of linked bullet points of the successful results during your project</li>
+
<p>Successes</p>
−
<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>
+
<li>We confirmed the deactivation of phytase due to heat shock.</li>
−
</ul>
+
<li>We found the improvement of thermostability by SAPs.</li>
 +
<li>Exposure of the His-tag due to SAPs is suggested.</li>
  
−
 
+
<p>Failures</p>
−
 
+
<li>We couldn't construct the control part (expression of phytase only) and couldn't assay the function of cysteine module.</li>
−
 
+
−
<div class="column half_size" >
+
−
 
+
−
<h5>Inspiration</h5>
+
−
<p>See how other teams presented their results.</p>
+
−
<ul>
+
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
 
</ul>
 
</ul>
  
−
</div>
 
  
  
  
 
</html>
 
</html>

Revision as of 03:37, 2 November 2017

Results

Until the Wiki freeze, we tried to construct plasmid with the insert we designed and managed to combine plasmid with INSERT 1, 2 or 3(we couldn't insert INSERT 4, which is a control of the INSERT 2 or 3).

To assay our construct, we cultured E. coli with IPTG induction, purified the crude extract by the His-tag and condensed them.

When the expressed protein (both INSERT 2 and 3) were cut at the signal peptides, the molecular weight becomes 52367.76 or 48575.69 respectively.

The figure bellow is the result of SDS-PAGE. We applied purified INSERT 2 (left) and INSERT 3 (right, not visible)and found that the band of INSERT 2 is at the correct place.

We assumed two possibility that explain why we couldn't get the band of INSERT 3; the concentration of protein is too small, the His-tag didn't interacted with the Nickel column and flowed through.

To determine the reason why we couldn't see the band, we conducted phytase activity assay with the crude extract, purified and condensed products, and the flow through of purification process.

As the table and figure above shows, we can see high phytase activity in the flow through and concluded that His-tag didn't interacted with the Nickel, probably because the His-tag was not exposed outside. Simultaneously, we can assume that self-Assembling Peptides (SAPs) helped His-tag being exposed and interacting with Nickel.

We applied crude extract of INSERT 2 (left) and INSERT 3. (right). The difference of mobility between INSERT 2 and 3 is very small but may be slightly different. (Difficult to judge)

Phytase thermostability

    We assayed thermostability of several kind of phytase;

    Though the degree of deactivation is different according to kind of phytase, the characteristic that remaining activity after 1 hour heat shock drops is common.

    From the comparison between INSERT 2 and 3, we can see that activity of phytase from insert 3 drops more drastically than that of from INSERT 2, suggesting that SAPs will improve thermostability of phytase.

Project Achievements

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.

    Successes

  • We confirmed the deactivation of phytase due to heat shock.
  • We found the improvement of thermostability by SAPs.
  • Exposure of the His-tag due to SAPs is suggested.

    • Failures

  • We couldn't construct the control part (expression of phytase only) and couldn't assay the function of cysteine module.