Difference between revisions of "Team:HKJS S/Results"

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<h1>Results</h1>
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<font size="7" color="blue">Results</font>
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<p>Here you can describe the results of your project and your future plans. </p>
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<p>Results are derived after 5 times of Gibson Assembly, 150 plasmid purification and 100 gel electrophoresis.</P>
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<img style="width:80%; height:auto; margin:auto;" src="https://static.igem.org/mediawiki/2018/7/7d/T--HKJS_S--equipments.png"/>
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<div class="center"><b>Equipment used</b></div>
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<p>Using The Inoue Method for Preparation and Transformation of Competent E. coli, all constructions are transformed into E. coli BL21 and Neb 5 alpha. Plasmid purification are done using the TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 and Monarch® Plasmid Miniprep Kit.pETBlue-2 is used for Blue-White screening. </p>
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<br/>
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<h1>Nitrogenase</h1>
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<p>With reference to Fig. A below, it is observed that in the 3rd lane after an hour of 42V gel electrophoresis, a comparably larger fragment is constructed using Gibson Assembly.</p>
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<img style="width:30%; height:auto; margin: auto;" src="https://static.igem.org/mediawiki/2018/6/62/T--HKJS_S--FigA.jpg"/>
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<div class="center"><b>Fig. A</b></div>
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<p>All constructs in Fig. B are comparably larger products assembled by Gibson Assembly. The circular DNA on the 5th lane to the right is plasmid purified Gibson assembly product and the 4th lane from the right is its linearized form using double cut EcoRI and PstI. This indicates that the product is Bio-Brick compatible. In addition, after several hours of gel electrophoresis with 42V, except for these two lanes mentioned above, all other bands migrate downwards to the 8kbp point. This concludes that among all constructs we have done, this product matches the most with our expectation.
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<img style="width:30%; height:auto; margin: auto;" src="https://static.igem.org/mediawiki/2018/5/50/T--HKJS_S--FigB.jpg"/>
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<div class="center"><b>Fig. B</b></div>
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<p>These shows that we have successfully assembled the nif cluster using the Gibson Assembly.</p>
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<br/>
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<h1>PET decomposition</h1>
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<p>As the high GC content make it difficult to complete PCR, we amplified the PETase and MHETase with PCR after codon optimization. For the reason that the band of MHETase is weak, we then try to perform gradient PCR using 6 different temperatures. After ligation and transformation, only PETase is successfully transformed. We express the protein and measured the size of it. With the help of HKUST team, the PETase is characterized using SDS-Page.</p>
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<img style="width:30%; height:auto; margin-left: 15%; margin-right:10%;;" src="https://static.igem.org/mediawiki/2018/e/ec/T--HKJS_S--SDS.jpg"/>
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<img style="width:30%; height:auto; margin-right:10%;;" src="https://static.igem.org/mediawiki/2018/2/2c/T--HKJS_S--PCR.jpg"/>
 
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<h3>What should this page contain?</h3>
 
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<li> Clearly and objectively describe the results of your work.</li>
 
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<li> Future plans for the project. </li>
 
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<li> Considerations for replicating the experiments. </li>
 
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<h3>Describe what your results mean </h3>
 
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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>
 
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<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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<h3> Project Achievements </h3>
 
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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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<ul>
 
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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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<div class="highlight decoration_A_full">
 
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<h3>Inspiration</h3>
 
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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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Revision as of 16:34, 17 October 2018

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JUDGING FORM ⇗
Results

Results are derived after 5 times of Gibson Assembly, 150 plasmid purification and 100 gel electrophoresis.

Equipment used

Using The Inoue Method for Preparation and Transformation of Competent E. coli, all constructions are transformed into E. coli BL21 and Neb 5 alpha. Plasmid purification are done using the TaKaRa MiniBEST Plasmid Purification Kit Ver.4.0 and Monarch® Plasmid Miniprep Kit.pETBlue-2 is used for Blue-White screening.


Nitrogenase

With reference to Fig. A below, it is observed that in the 3rd lane after an hour of 42V gel electrophoresis, a comparably larger fragment is constructed using Gibson Assembly.

Fig. A

All constructs in Fig. B are comparably larger products assembled by Gibson Assembly. The circular DNA on the 5th lane to the right is plasmid purified Gibson assembly product and the 4th lane from the right is its linearized form using double cut EcoRI and PstI. This indicates that the product is Bio-Brick compatible. In addition, after several hours of gel electrophoresis with 42V, except for these two lanes mentioned above, all other bands migrate downwards to the 8kbp point. This concludes that among all constructs we have done, this product matches the most with our expectation.

Fig. B

These shows that we have successfully assembled the nif cluster using the Gibson Assembly.


PET decomposition

As the high GC content make it difficult to complete PCR, we amplified the PETase and MHETase with PCR after codon optimization. For the reason that the band of MHETase is weak, we then try to perform gradient PCR using 6 different temperatures. After ligation and transformation, only PETase is successfully transformed. We express the protein and measured the size of it. With the help of HKUST team, the PETase is characterized using SDS-Page.