<li>Clearly and objectively describe the results of your work.</li>
<li>Future plans for the project.</li>
<li>Considerations for replicating the experiments.</li>
</ul>
</div>
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<h2>Describe what your results mean</h2>
<hr>
<ul>
<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>
<li>Show data, but remember <b>all measurement and characterization data must also be on the Part's Main
Page on the <ahref="http://parts.igem.org/Main_Page">Registry</a>.</b> Otherwise these data will
not be in consideration for any medals or part awards!
</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>
</ul>
</div>
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<h1>Phasin Secretion</h1>
<p>After several iterations and modifications to our experimental designs, we utilized a GFP marker on our phasin proteins to detect intracellular and extracellular concentrations through fluorescence reading on a 96 well plate reader. It is important to note that we chose not to interpret the exact quantifications of concentrations between our intracellular and extracellular results due to the differences in procedural dilutions. For example, the extracellular components were directed separated and sampled after centrifugation. Meanwhile, the intracellular components were processed via cell lysis first, having been diluted at different amounts. Instead, we have compared these values in relation to each other through comparisons.
</p>
<h3>The results communicated the following:
</h3>
<ol>
<li>In both extracellular and intracellular samples, the negative control group had minimal fluorescence readings verifying the lack of GFP-tagged phasin production in untransformed E. Coli.
</li>
<li>In the extracellular (supernatant) samples, the type I HlyA secretion system had the highest fluorescence values followed by the non secretion system, the VNp6 secretion system, and type II TorA secretion system.
</li>
<li>In the intracellular samples, the type I HlyA system had the highest fluorescence values followed by the non secretion system, the VNp6 secretion system, and the type II TorA secretion system.
<p>Our readings presented us with fluorescent measurements for the nonsecretion group that was higher than that of TorA and VNp6 in the extracellular media. A possible explanation for these unexpected results may be due to the hindrance of phasin-GFP fusion protein production in the presence of a type II or VNp secretion system in combination with unintended leakage from the intracellular components of the E. coli during our separation procedures. Future investigations should investigate an improved separation technique.</p>
</div>
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<h1>PHB Production</h1>
<p>Before moving on to designing our PHB secretion system, we wanted to confirm our selected from model for PHB production in E. coli. Our system is based on part Part:BBa_K934001 from Tokyo Tech 2012. The team used gas chromatography and mass spectrometry (GC/ MS) in order to identify the products. Other past teams have also utilized chloroform in order to attain PHB. However, with our limited resources, we found an alternative to these methods by verifying PHB production through staining and microscopy.</p>
<p>We followed a simple Black Sudan B staining protocol to prepare slides of our experimental PHB-producing E. coli cells and untransformed E. coli as our negative control for comparison.</p>
<p>Following the success of our PHB production model, we moved forward by integrated PHB production with the earlier secretion models (more information can be found in Engineering Success). After incubating our E. coli cells for PHB production in a 3% glucose media, we quantified the extracellular and intracellular concentrations of PHB through absorbance readings on the 96 well plate reader. Additionally, we verified these results through a second fluorescence reading to confirm a correlation between phasin and PHB production with concentrations of GFP.</p>
<p>The results communicated the following (see Results for more)</p>
<ol>
<li>In both intracellular and extracellular samples, the negative control group had minimal fluorescence readings verifying the absence of GFP-tagged phasin production.</li>
<li>In the extracellular (supernatant) samples, the VNp6 system had the highest fluorescence values followed by the non secretion system, the HlyA system, the TorA system, and the negative control. The non secretion system had the highest absorbance values followed by the negative control, the HlyA system, the TorA system, and the VNp6 system.
</li>
<li>In the intracellular samples, the VNp6 system had the highest fluorescence values, followed by the non secretion system, the HlyA system, the TorA system, and the negative control. The negative control and HlyA had the highest absorbance values, the non-secretion system, the VNp6 system, and the TorA system.</li>
</ol>
<imgsrc="">
<p>We realized our chosen method of PHA quantification is restricted in accuracy and reliability. For future reference, in order to optimize the accuracy of PHA determinations with spectrophotometric quantification, we learnt that a calibration curve comparing known PHA values and measured absorbance values of Sudan Black B dilutions is a necessary component for quantifying PHA values. We suggest following the improved methodology proposed by Porras et al. However, if possible, we recommend future teams use techniques such as HPLC, gas chromatography, or flow cytrometry to obtain results.</p>
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<divclass="col-lg-8">
<h2>Project Achievements</h2>
<hr>
<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>
<li>A list of linked bullet points of the successful results during your project</li>
<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
