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− | {{IGEM_TopBar}}
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− | {{Fujian_United}}
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| <html> | | <html> |
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− | <div class="column full_size"> | + | <link rel="stylesheet" type="text/css" |
− | <h1>Results</h1>
| + | href="https://2021.igem.org/wiki/index.php?title=Template:Fujian_United/StyleCSS&action=raw&ctype=text/css"/> |
− | <p>You can describe the results of your project and your future plans here. </p>
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− | </div>
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| + | <!--Team--> |
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| + | <a href="https://2021.igem.org/Team:Fujian_United/Team">Team</a> |
| + | <div class="child-nav"> |
| + | <ul class="child-list"> |
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| + | <a href="https://2021.igem.org/Team:Fujian_United/Team">Team Members</a> |
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| + | <a href="https://2021.igem.org/Team:Fujian_United/Attributions">Attributions</a> |
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| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Collaborations">Collaboration</a> |
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| + | </ul> |
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− | <h3>What should this page contain?</h3> | + | <!--Modeling--> |
− | <ul>
| + | <li class="item"> |
− | <li> Clearly and objectively describe the results of your work.</li> | + | <a href="https://2021.igem.org/Team:Fujian_United/Model">Modeling</a> |
− | <li> Future plans for the project. </li> | + | </li> |
− | <li> Considerations for replicating the experiments. </li>
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− | </ul> | + | |
− | </div> | + | |
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| + | <!--Entrepreneurship--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Entrepreneurship">Entrepreneurship</a> |
| + | </li> |
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| + | <!--Implementation--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Implementation">Implementation</a> |
| + | </li> |
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| + | <!--Human Practice--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Human_Practices">Human Practice</a> |
| + | <div class="child-nav"> |
| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Human_Practices">Integrated Human |
| + | Practice</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Communication">Communication</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Fundraising">Fundraising</a> |
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| + | </ul> |
| + | </div> |
| + | </li> |
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− | <div class="column two_thirds_size" > | + | <!--Parts--> |
− | <h3>Describe what your results mean </h3> | + | <li class="item"> |
− | <ul> | + | <a href="https://2021.igem.org/Team:Fujian_United/Parts">Parts</a> |
− | <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> | + | <div class="child-nav"> |
− | <li> Show data, but remember <b>all measurement and characterization data must also be on the Part's Main Page on the <a href="http://parts.igem.org/Main_Page">Registry</a>.</b> Otherwise these data will not be in consideration for any medals or part awards! </li> | + | <ul class="child-list"> |
− | <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>
| + | <li class="child-item"> |
− | </ul> | + | <a href="https://2021.igem.org/Team:Fujian_United/Parts">Parts |
− | </div> | + | Collection</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Engineering">Engineering</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
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| + | <!--Project--> |
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| + | <a href="https://2021.igem.org/Team:Fujian_United/Description">Project</a> |
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| + | <ul class="child-list"> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Description">Description</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Experiments">Experiments</a> |
| + | </li> |
| + | <li class="child-item active"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Results">Results</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Proof_Of_Concept">Proof of Concept</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Notebook">Notebook</a> |
| + | </li> |
| + | <li class="child-item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United/Safety">Safety</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
| + | </li> |
| | | |
− | <div class="clear extra_space"></div> | + | <!--Home--> |
| + | <li class="item"> |
| + | <a href="https://2021.igem.org/Team:Fujian_United">Home</a> |
| + | </li> |
| + | </ul> |
| + | </div> |
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| + | <!--图片--> |
| + | <div class="page-header"> |
| + | <img src="https://static.igem.org/mediawiki/2021/9/94/T--Fujian_United--bg_1.png" alt=""> |
| + | </div> |
| | | |
| + | <!--内容--> |
| + | <div class="content-middle-outer"> |
| + | <div class="content-middle"> |
| + | <section class="article p-t-30 p-b-54"> |
| + | <h1 class="content-header">Results</h1> |
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− | <div class="column two_thirds_size" > | + | <section class="en-indent-all"> |
− | <h3> Project Achievements </h3> | + | <h1 class="title">1. Glucoamylase expression plasmids construction</h1> |
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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> | + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/8/8e/T--Fujian_United--img_results_1.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig.1 Glucoamylase expression plasmids in this project</span> |
| + | </div> |
| + | <p>Glucoamylase (GA) is one of the key enzymes during the starch to glucose process. A genetically |
| + | engineered GA expressing <i>Saccharomyces cerevisiae</i> strain is strongly required to save the |
| + | enzyme cost in starch based bioethanol industry.</p> |
| + | <p>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> |
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− | <ul> | + | <div class="img-container m-t-18"> |
− | <li>A list of linked bullet points of the successful results during your project</li> | + | <img src="https://static.igem.org/mediawiki/2021/2/25/T--Fujian_United--img_results_2.jpg" alt="" style="width: 60%"> |
− | <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> | + | <span class="figure">Fig. 2 Construction of Saccharomycopsis fibuligera derived glucoamylase expressing plasmids</span> |
− | </ul> | + | </div> |
| + | <p>The Golden gate cloning method was used to prepare the GA-expressing plasmids. The basic parts of the |
| + | plasmids such as the pYES2 backbone, GA coding sequence, promoters, and terminators were all |
| + | amplified successfully firstly (Fig. 2 A and B), then the multiplex GA expression cassettes were |
| + | assembled using the overlap PCR method, the resulting parts were cut with <i>Sap</i>I restriction |
| + | enzyme and ligated with the backbone, which was cut with the same enzyme. The primer pairs |
| + | YZ-IGEM-1/CYC1t-R were set to verify the positive colonies (Fig. 2C). We chose 24 colonies to verify |
| + | whether the plasmids were correct or not using the colony PCR, the positive rate for the plasmids |
| + | pYES2-TGC, pYES2-HGC, and pYES2-IGC were 11/24, 17/24, and 17/24, respectively. Two or three |
| + | positive colonies were sequenced to verify further. </p> |
| + | </section> |
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− | </div> | + | <section class="en-indent-all"> |
| + | <h1 class="title">2. GA plasmids transformation</h1> |
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| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/3/35/T--Fujian_United--img_results_3.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig. 3 GA-expressing plasmids transformation and positive <i>S. cerevisiae</i> transformants selection using 50 μg/mL (A) and 350 μg/mL (B) hygromycin. Top left, pYES2-ctl. Top right, pYES2-TGC. Bottom left, pYES2-HGC. Bottom right, pYES2-IGC. </span> |
| + | </div> |
| + | <p>The constructed GA-expressing plasmids were chemically transformed into the <i>S. cerevisiae</i> |
| + | CEN.PK2-1C strain, the positive transformants were selected against a relatively low concentration |
| + | of hygromycin (50 μg/mL) firstly to ensure colony growth. As shown in Fig. 1A, plasmids pYES2-ctl, |
| + | pYES2-TGC, pYES2-HGC, and pYES2-IGC seemed all transformed into the CEN.PK2-1C strain. Then, we |
| + | streaked colonies from the low concentration hygromycin plates onto the high concentration |
| + | hygromycin (350 μg/mL) plates again to verify the positive transformants. </p> |
| | | |
| + | <br> |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/b/b3/T--Fujian_United--img_results_4.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig. 4 Verification of the plasmids pYES2-ctl (A), pYES2-TGC (B), pYES2-HGC and pYES2-IGC (C) transformation via colony PCR in CEN.PK2-1C strain. </span> |
| + | </div> |
| + | <p>The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to |
| + | verify the plasmids transformation again. From Fig. 4 we can see that positive bands implied the |
| + | plasmids transformation successfully. </p> |
| + | </section> |
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− | <div class="column third_size" > | + | <section> |
− | <div class="highlight decoration_A_full"> | + | <h1 class="title">Glucoamylase activity assay</h1> |
− | <h3>Inspiration</h3>
| + | |
− | <p>See how other teams presented their results.</p>
| + | |
− | <ul>
| + | |
− | <li><a href="https://2019.igem.org/Team:Newcastle/Results">2019 Newcastle</a></li>
| + | |
− | <li><a href="https://2019.igem.org/Team:Munich/Results">2019 Munich </a></li>
| + | |
− | <li><a href="https://2019.igem.org/Team:Tec-Chihuahua/Results">2019 Tec Chihuahua</a></li>
| + | |
− | <li><a href="https://2020.igem.org/Team:Aalto-Helsinki/Results">2020 Aalto Helsinki</a></li>
| + | |
− | <li><a href="https://2020.igem.org/Team:GreatBay_SCIE/Results">2020 GreatBay SCIE</a></li>
| + | |
− | <li><a href="https://2020.igem.org/Team:Queens_Canada/Results">2020 Queens Canada</a></li>
| + | |
− | | + | |
− | </ul>
| + | |
− | </div>
| + | |
− | </div> | + | |
| | | |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/7/7f/T--Fujian_United--img_results_5.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig. 5 <i>S. cerevisiae</i> strains harboring various plasmids glucoamylase activity determination. **Statistical significance between indicated strains, p < 0.01. ns, not significant. Data represent the means of two independent colonies.</span> |
| + | </div> |
| | | |
| + | <p>The GA activity of <i>S. cerevisiae</i> strains harboring various plasmids was determined using the |
| + | glucoamylase activity assay kit. As shown in Fig. 5, the control stain (left column) exhibited no GA |
| + | activity. 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. However, 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> |
| + | </section> |
| | | |
| + | <section class="en-indent-all"> |
| + | <h1 class="title">Fermentation test</h1> |
| | | |
| + | <div class="img-container m-t-18"> |
| + | <img src="https://static.igem.org/mediawiki/2021/6/61/T--Fujian_United--img_results_6.jpg" alt="" style="width: 60%"> |
| + | <span class="figure">Fig. 6 Glucose concentration inside the cell during the GA-expressing <i>S. cerevisiae</i> strains corn starch fermentation process.</span> |
| + | </div> |
| + | <p>To verify the GA secretion capacity of the GA-expressing <i>S. cerevisiae</i> strains, we measured the glucose concentration inside the cell during the corn starch fermentation process. As shown in Fig. 6A, 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. 6B), 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. In contrast to this, when the GA was added in the medium preparation process, all the strains showed almost the same glucose concentration at 24 h. Overall, when the GA was removed from the medium preparation, the GA-expressing strains showed a comparable glucose production capacity with the GA addition situation. </p> |
| + | </section> |
| + | </section> |
| + | </div> |
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