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<h2> Our Solution </h2>

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<h4>Enzyme Analysis</h4>
<p>Once the devices were transformed into the yeast cells and the expression verified, it was planned that a series of experiments done to determine the effectiveness of the different lignin degrading and helper enzymes in combination. Given that this point was not reached during our time this year, the following section contains the plans for what was to happen if that point was reached. </p>

<p>As the devices were designed to export the enzymes from the cells, a simple centrifuge and extraction of supernatant could provide a solution containing the target enzyme. This amount would be quantified using a Nanodrop. The solution would have then been used in a simple lignin-degradation assay and a given amount of lignin degraded per mol of enzyme for each individual enzyme could be determined. </p>

<p>After this baseline is established, the enzymes would then be tested in combination using a Design of Experiments set-up. Using linear regression, a model could be developed to calculate amount of lignin broken down per amount of each enzyme present. </p>
<img src="https://static.igem.org/mediawiki/2015/c/c3/Purdue_1st_equation.png" alt="Equation to calcuate totla lignin broken down per amount of each enzyme." style="width:494px;height:48px;">
<p>Interactions between enzymes, both positive and negative, could be determined and the use of statistical optimization could eliminate enzymes and interactions that have little effect, leading to the isolation of useful enzymes and a model that predicts the amount of lignin degraded. </p>
<img src="https://static.igem.org/mediawiki/2015/d/d8/Purdue_2nd_equation.png" alt="Equation accounting for the interaction of enzymes." style="width:625px;height:40px;">
<div>This model could then be used to estimate the amount of enzymes needed for a full-scale bioreactor and follow with the size of yeast culture needed to produce the required amount of enzyme. </div>

<p>Additional assays used to determine the lifetime of expressed enzymes could then be factored into the model, enabling it to account for the natural degradation of enzymes over time, providing a better estimate of the size of yeast culture required for a large-scale bioreactor. </p>

<h4>Sources</h4>
<ul>
<li>https://www.neb.com/products/e0546-biobrick-assembly-kit</li>
<li>Agu, Chukwuma, Klein, Reinhard,  Lengler, Johannes, et al. “Bacteriophage-encoded toxins: the l-holin protein causes caspase-independent non-apoptotic cell death of eukaryotic cells.” Cellular Microbiology 9.7 (2007): 1753–1765. Online.</li>
<li>"Ethanol Prodution." ICM. n.p., n.d. Web. 18 September 2015.</li>
<li>"Role of Yeast in Production of Alcoholic Beverages." Hawaii Botany 135. n.p., n.d. Web. 18 September 2015.</li>
/<ul>

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<h4>Kill Switch/Containment</h4>
<p>We decided to use parts already in the iGEM registry for our killswitch construct. We chose an oxygen repressible promoter(Part:BBa_K950002), a kozak sequence(BBa_J63003), a lysis sequence(BBa_K809605), and a terminator(BBa_K801012) all optimized for yeast from the registry. A kozak sequence is a ribosome binding site for eukaryotic organisms. The lysis sequence is also known as Lambda Holin, a protein from a bacteriophage that has also been shown to be cytotoxic in eukaryotic cells (Agu 1753). We then pieced these parts together using 3A assembly. First we combined the promoter and kozak and the coding sequence and terminator, then all four to make a full device. After that we used standard assembly to insert the sequence into a yeast vector(pSB1C3), so it was ready to be transformed into our yeast chassis.</p>
<h4>A Assembly</h4>


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<h4>Prototype</h4>
<p>With current bioreactor design in mind, our yeast will not require any intricate conditions. In place of the pretreatment processes normally used, our yeast would only require to be added into a mixer/container with the lignocellulosic biomass and an adequate amount of sugar for nutrients. With the relative ease to use our synthetic yeast, the prototype was easy to develop and design. We simply used glass mason jars as containers and attached a clothes hanger to an electric-drill to change it’s function and allow it to be used as a mixer. </p>

<p>Our pretreatment process would involve two steps: a lignin digestion phase, a liquefaction phase, and would also be limited to only being utilized with batch-reactors. Our process would be limited to batch-reactors because if our process was applied to industries there would have to be filtration phases to remove the yeast after step one, and the cellulase and xylanases in step two. If the yeast is not removed after step one, there would be competition between our synthetic yeast and J4, which would decrease J4’s effectiveness. And if the cellulases and xylanases are not removed after step two, this would slightly decrease the efficiency of J4 by decreasing the overall concentration of biomass in the solution.</p>
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<h4>Materials</h4>
<ul>
<li>Electric Drill</li>
<li>Rubber Bands</li>
<li>Glass Mason Jars</li>
<li>Wire Clothes Hanger</li>
<li>Synthetic Yeast</li>
<li>.5g Cellulase</li>
<li>.5g Xylanase</li>
<li>1g Switch Grass</li>
<li>10L Water</li>
</ul>
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<h4>Method</h4>
<ol>
<li>First, add the synthetic yeast, biomass, sugar, and water into the glass mason jar. </li>
<li>Make a hole, just large enough to fit the clothes hanger in.</li>
<li>Attach the clothes hanger to the power drill and make cut the clothes hanger as so:</li>
<img src="https://static.igem.org/mediawiki/2015/4/4f/Purdue_Drill_with_crowbar.png" alt="Cell Wall Diagram" style="width:551px;height:281px;">
<li>Attach the lid to the jar and set the drill to a slow setting with rubber bands holding the trigger in place.</li>
<li>Wait for 24 hours.</li>
<li>Move biomass into 2nd glass mason jar.</li>
<li>Add cellulase and xylanase.</li>
<li>Attach the lid to the jar and set the drill to a slow setting with rubber bands holding the trigger in place.</li>
<li>Wait for 24 hours</li>
<li>The biomass is now ready to undergo fermentation.</li>
</ol>
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<p>All in all, our prototype was developed with tools found inside of our lab. Even though our design may not be efficient, we can still collect sufficient data through multiple iterations, as long as we follow the same procedures. We plan on utilizing this design to collect data in the future and hopefully measure the efficiency of our synthetic yeast’s capability to digest lignin.</p>

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<h4>Sources</h4>
<ul>
<li>http://cdn.toptenreviews.com/rev/misc/articles/8925/5-creative-ways-8.jpg</li>
</ul>
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