Difference between revisions of "Team:Purdue/ProjectDevelopment"

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<h1>Enzyme Analysis</h1>
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<h2>Project Development</h2>
  
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<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>
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<p>The Biomakers started their project development from a human practices perspective, thinking of five broad areas to search for problems in: energy, food, waste, water and livestock. Ideas panned out from each of these categories but one that garnered a lot of interest was the use of biodigesters as a means of agricultural and municipal waste management.</p>  
  
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<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>
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<p>The focus then shifted to assessing the major issues within biodigesters. After learning about the benefits of codigestion, it was decided to focus in one the major issue with dealing with plants in biodigestion: the difficulty of lignin degradation.</p>
  
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<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>
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<p>The search for lignin-degrading solutions ended in a meeting with Dr. Michael Schart (Department of Entomology, Purdue University). Dr. Scharf’s research revolves around the identification of lignin-degrading enzymes in termites. The project idea began to form from this meeting: a synthetic yeast system that produces and secretes lignin-degrading enzymes as an enzymatic alternative for thermal pretreatment. </p>
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<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;">
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<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>
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<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;">
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<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>
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<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>
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<p>Although the termite enzymes were strong candidates, the search was widened for any woody-plant-eating species and their lignin-degrading enzymes. With a wide field, design criteria were set for enzymes that operate at neutral pH and room temperatures, conditions that would make the project more feasible and easier to test. These criteria narrowed the field to genes from both termites and various white rot fungi. </p>
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<h4>Prototype</h4>
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<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>
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<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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<p>At this point, the idea seemed to be coming along fairly well until we met with Dr. Nate Mosier (Department of Agricultural and Biological Engineering, Purdue University), who gave some perspective on why enzymatic pretreatment is so difficult: the byproducts of lignin degradation inhibit cellulase enzymes, so degradation of lignin polymers can actually and has been proven to lower the conversion rate of cellulose into ethanol. This new information provided a major roadblock, one which was thought to be an end to a viable project based on synthesizing lignin-degrading enzymes, when a significant piece of work in the literature arose in the search. </p>
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<h4>Materials</h4>
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<p>The J4 yeast, co-developed by synthetic biologists at University of Illinois, University of California Berkeley, and a few others, is a strain of synthetic yeast that internalizes the conversion of cellulose into ethanol from the stage of cellobiose (cellulose disaccharide) to the final stage of ethanol. The internalization meant that, if the lignin was degraded in the exterior of the cell, the byproducts could not interfere with the enzyme activity going on inside the cell: the project was back on track. </p>
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<li>Electric Drill</li>
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<p>This discovery refined the project and contextualized the end goal of creating a strain of synthetic yeast that would complement the J4 strain, filling in the last piece of the puzzle from woody material to useful biofuel. This would complete the picture of a simplified biofuel conversion process that is based on only one easily engineered species, rather than depending on the natural host of organisms found in current biodigesters. This simple yet easily modified platform would then open up new possibilities for biofuel conversion optimization, fulfilling the mission of making biofuels a more viable energy alternative. </p>
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<li>Rubber Bands</li>
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<li>Glass Mason Jars</li>
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<li>Wire Clothes Hanger</li>
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<li>Synthetic Yeast</li>
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<li>.5g Cellulase</li>
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<li>.5g Xylanase</li>
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<li>1g Switch Grass</li>
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<li>10L Water</li>
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<h4>Method</h4>
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<ol>
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<li>First, add the synthetic yeast, biomass, sugar, and water into the glass mason jar. </li>
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<li>Make a hole, just large enough to fit the clothes hanger in.</li>
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<li>Attach the clothes hanger to the power drill and make cut the clothes hanger as so:</li>
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<img src="https://static.igem.org/mediawiki/2015/4/4f/Purdue_Drill_with_crowbar.png" alt="Cell Wall Diagram" style="width:551px;height:281px;">
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<li>Attach the lid to the jar and set the drill to a slow setting with rubber bands holding the trigger in place.</li>
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<li>Wait for 24 hours.</li>
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<li>Move biomass into 2nd glass mason jar.</li>
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<li>Add cellulase and xylanase.</li>
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<li>Attach the lid to the jar and set the drill to a slow setting with rubber bands holding the trigger in place.</li>
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<li>Wait for 24 hours</li>
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<li>The biomass is now ready to undergo fermentation.</li>
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</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>
 
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<li>http://cdn.toptenreviews.com/rev/misc/articles/8925/5-creative-ways-8.jpg</li>
 
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Latest revision as of 03:23, 19 September 2015

Team Banner

Project Development

The Biomakers started their project development from a human practices perspective, thinking of five broad areas to search for problems in: energy, food, waste, water and livestock. Ideas panned out from each of these categories but one that garnered a lot of interest was the use of biodigesters as a means of agricultural and municipal waste management.

The focus then shifted to assessing the major issues within biodigesters. After learning about the benefits of codigestion, it was decided to focus in one the major issue with dealing with plants in biodigestion: the difficulty of lignin degradation.

The search for lignin-degrading solutions ended in a meeting with Dr. Michael Schart (Department of Entomology, Purdue University). Dr. Scharf’s research revolves around the identification of lignin-degrading enzymes in termites. The project idea began to form from this meeting: a synthetic yeast system that produces and secretes lignin-degrading enzymes as an enzymatic alternative for thermal pretreatment.

Although the termite enzymes were strong candidates, the search was widened for any woody-plant-eating species and their lignin-degrading enzymes. With a wide field, design criteria were set for enzymes that operate at neutral pH and room temperatures, conditions that would make the project more feasible and easier to test. These criteria narrowed the field to genes from both termites and various white rot fungi.

At this point, the idea seemed to be coming along fairly well until we met with Dr. Nate Mosier (Department of Agricultural and Biological Engineering, Purdue University), who gave some perspective on why enzymatic pretreatment is so difficult: the byproducts of lignin degradation inhibit cellulase enzymes, so degradation of lignin polymers can actually and has been proven to lower the conversion rate of cellulose into ethanol. This new information provided a major roadblock, one which was thought to be an end to a viable project based on synthesizing lignin-degrading enzymes, when a significant piece of work in the literature arose in the search.

The J4 yeast, co-developed by synthetic biologists at University of Illinois, University of California Berkeley, and a few others, is a strain of synthetic yeast that internalizes the conversion of cellulose into ethanol from the stage of cellobiose (cellulose disaccharide) to the final stage of ethanol. The internalization meant that, if the lignin was degraded in the exterior of the cell, the byproducts could not interfere with the enzyme activity going on inside the cell: the project was back on track.

This discovery refined the project and contextualized the end goal of creating a strain of synthetic yeast that would complement the J4 strain, filling in the last piece of the puzzle from woody material to useful biofuel. This would complete the picture of a simplified biofuel conversion process that is based on only one easily engineered species, rather than depending on the natural host of organisms found in current biodigesters. This simple yet easily modified platform would then open up new possibilities for biofuel conversion optimization, fulfilling the mission of making biofuels a more viable energy alternative.