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| + | <section class="article p-t-30 p-b-54"> |
| + | <h1 class="content-header">Proof of Concept</h1> |
| + | |
| + | <section class="en-indent-all"> |
| + | <h1 class="title">Overview</h1> |
| + | |
| + | <p>Glucoamylase (GA) is one of the key enzymes during the starch to glucose process. Our product is |
| + | Glucoamylase-in Saccharomyces cerevisiae. With it, the ethanol manufacturer can reduce a great |
| + | amount of glucoamylase input, thus, lower the cost and increase efficiency. To make our product, we |
| + | design to construct a plasmid that expresses the gene of secreting glucoamylase, and insert it into |
| + | the Saccharomyces cerevisiae. While reducing the additional glucoamylase needed during ethanol |
| + | production, the fermentation performance stays the same. In terms of constructing the plasmid, To |
| + | express the glucoamylase gene under different strength promoters, we designed three plasmids |
| + | together with a control plasmid pYES2-ctl (Fig. 1A), that is, GA is expressed under constitutive |
| + | promoter TEF1 (Fig. 1B), GA is expressed under glucose inducible promoter HXT7 (Fig. 1C), and GA is |
| + | expressed under glucose repression promoter ICL1 (Fig. 1D).</p> |
| + | <p>To assess the performance of the Saccharomyces cerevisiae with our constructed plasmids, we carried |
| + | out different tests. Firstly, we tested the enzymatic activity of the glucoamylase the Saccharomyces |
| + | cerevisiae secreted by the glucoamylase activity assay kit and the fermentation tests were also |
| + | conducted to verify the GA secretion capacity by measuring the yielded glucose and ethanol.</p> |
| + | |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/b/b3/T--Fujian_United--ing_proof_of_concept_1.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig.1 Glucoamylase expression plasmids in this project</span> |
| + | </div> |
| + | </section> |
| + | |
| + | <section> |
| + | <h1 class="title">Experimental Support</h1> |
| + | |
| + | <ul class="normal"> |
| + | <li> |
| + | <p>Glucoamylase activity</p> |
| + | <p>Since we have obtained the Glucoamylase-in Saccharomyces cerevisiae harboring various |
| + | plasmids, we need to ensure that our strains could perform the expected. To verify the |
| + | glucoamylase activity, we used the glucoamylase activity assay kit and the result is given |
| + | below.</p> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/0/0a/T--Fujian_United--ing_proof_of_concept_2.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig 2. <i>S. cerevisiae</i> strains harboring various plasmids glucoamylase activity determination.</span> |
| + | </div> |
| + | <p>Though the strain in which the expression of GA was under the glucose repressive promoter |
| + | ICL1 exhibited a very low GA activity, this is because the substrate of the glucoamylase |
| + | assay kit is glucose, the expression of the GA cassette was inhibited.</p> |
| + | <p>The strains harboring pYES2-TGC and pYES2-HGC plasmids, which means the GA’s expression was |
| + | driven by the constitutive promoter TEF1 and glucose inducible promoter HXT7, showed almost |
| + | the same and high GA activity.</p> |
| + | </li> |
| + | <li> |
| + | <p>Fermentation performance</p> |
| + | <p>Considering the potential of our Glucoamylase-in Saccharomyces cerevisiae to be applied in |
| + | the alcohol industry, we decided to conduct several mini fermentation processes and analyze |
| + | their fermentation performance by measuring the amount of glucose and alcohol.</p> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/2/21/T--Fujian_United--ing_proof_of_concept_3.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig 3. Glucose concentration inside the cell during the GA-expressing <i>S. cerevisiae</i> strains corn starch fermentation process.</span> |
| + | </div> |
| + | <p>As shown above, at the initial stage (0 h), when the GA was added during the |
| + | “starch-to-glucose” process, higher contents of glucose were detected than the process |
| + | without GA addition. This is because GA can degrade starch to make more glucose. Along with |
| + | the fermentation process, the glucose concentration in the pYES2-ctl containing strain |
| + | decreased obviously (Fig. 3B), due to the without more glucose production, glucose was |
| + | utilized by the strain. However, the pYES2-TGC and pYES2-HGC containing stains showed higher |
| + | glucose concentration without GA addition, this is because the GA produced by the strains |
| + | could hydrolysis starch to prepare glucose, in other words, the GA-expressing strains could |
| + | reduce the enzyme usage.</p> |
| + | |
| + | <br> |
| + | <div class="img-container"> |
| + | <img src="https://static.igem.org/mediawiki/2021/c/cf/T--Fujian_United--ing_proof_of_concept_4.jpg" alt=""> |
| + | <span class="figure">Fig. 4 Alcohol production ability of 4 glucoamylase-in Saccharomyces cerevisiae against enzyme concentration and fermentation hours</span> |
| + | </div> |
| + | <p>We built the model to predict the optimal condition for alcohol production of 4 |
| + | glucoamylase-in Saccharomyces cerevisiae (Fig. 4). Below are the conclusions:</p> |
| + | |
| + | <br> |
| + | <section class="en-indent-all"> |
| + | <p>For A strain, the highest value of alcohol concentration is 0.1993 g/L when enzyme |
| + | concentration is given 0 and the fermentation time is 24 hours;</p> |
| + | <p>For B strain, the highest value of alcohol concentration is 0.1991 g/L when enzyme |
| + | concentration is given 0.01 and the fermentation time is 22 hours;</p> |
| + | <p>For C strain, the highest value of alcohol concentration is 0.2049 g/L when enzyme |
| + | concentration is given 0 and the fermentation time is 13.5 hours;</p> |
| + | <p>For D strain, the highest value of alcohol concentration is 0.2018 g/L when enzyme |
| + | concentration is given 0.56 and the fermentation time is 13 hours.</p> |
| + | </section> |
| + | <br> |
| + | <p>Overall consideration, we strongly recommend yeast C for industrial alcohol production which |
| + | requires fewer fermentation hours, less additional enzyme but higher yield.</p> |
| + | </li> |
| + | </ul> |
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