Difference between revisions of "Team:ShanghaitechChina/Description"

 
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<a href="#p1">Overview</a>
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<a href="#p1"><h5>Improve the characterization</h5></a>
 
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<a href="#p2"><h5>Optimize the codon</h5></a>
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Thinking beyond laboratory is one of the key factors that would affect the yield of scientific researches into real social interests. Thankfully, this idea has been recognized by the iGEM community for a very long time. Not only should every team participate in various social activities, but also we need to keep a real time interaction with the society, the place that we ultimately hope to create significance.<p></p>
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This year, we have done the two improvement work:<p></p>
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This year, our team applied the knowledge of synthetic biology to seek feasible solutions to the energy issue. Urgent as it is, the energy issue is more than a regional problem, but has raised global concerns. In this social context, converting solar energy into chemical energy is undoubtedly of fundamental and industrial importance. Our original interest on this topic was initially sparked by recent cutting-edge research in artificial photosynthesis. To enhance our understandings about energy issues, and meanwhile, arouse public awareness of synthetic biology, we raised multiple public questionnaires and dialogues within academe and industry. In communications with industrial community, field experts as well as administrative authority afterwards, we realized that development of a robust, sustainable and scalable approach for hydrogen production would be socially relevant, industrially important, but technically challenging. This has led us to set our ultimate goals to solve such challenge by leveraging the power of synthetic biology. We eventually proposed and demonstrated a sun-powered biofilm-interfaced artificial hydrogen-producing system, Solar Hunter. Benefiting from the real time social interactions, we kept modifying the system to be more and more adaptable to the scalable industrial application. This new system will in return, introduce a synthetic biology-based approach towards energy applications in industrial context.<p></p>
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1. We improved the characterization of biobrick: <a href="http://parts.igem.org/Part:BBa_K1583003">BBa_K1583003</a> which was originally characterized by iGEM15_TU_Delft.<p></p>
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Additionally, while conducting our research with the interaction in the social context, our team also actively participated in a variety of educational propagation and public engagements. As the first-ever iGEM team at ShanghaiTech, this year we established the iGEM Day for Public Interests (iDPI), where we held a Lab Open House Day for the visiting high school student from Shanghai Coordinate International School, aiming at arousing their interests in science, especially in synthetic biology. Meanwhile, we introduced the iGEM to them as well, and are now helping them to build their first-ever high school team. Besides, our team collaborated with another two iGEM teams in China, one at Jiangsu Normal University, and the other at Tsinghua University.<p></p>
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2. We optimized the codons for a major functional part of Hydrogenase, HydA. This biobrick:<a href="http://parts.igem.org/Part:BBa_K535002">BBa_K535002</a> was originally designed by: iGEM11_UNAM-Genomics_ Mexico. <p></p>
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<a href="https://2016.igem.org/Team:ShanghaitechChina/HP/Gold">Click here to find how we Integrated Human Practice.</a>
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In the following content, we introduce our work in detail to illustrate why we think we met the criteria.<p></p><p></p>
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          <h1 align="center">Bridge the Gap Between the Academics and Real Social Applications</h1>
 
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          <h2 align="center">Social Research on the Energy Issue</h2>
 
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As previously addressed, the importance and urgency to seek a feasible solution to the energy issue is undebatable. The energy issue is neither just a regional nor national problem, but instead a global one. In response to the urgent need, vast efforts and time have already been devoted to those relevant studies, however, only few of which is really able to be put into real applications. Actually, the biggest obscure restraining the feasible solution to alleviate the energy stress is mainly due to the huge gap between the research results and the massive industrialization. In another word, not only should we emphasize the technological advancements, but also we ought to raise the strong awareness to integrate the scientific results into real industrial application to yield concrete social values.<p></p>
 
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To better understand the energy issue from a border aspect, our team carried out a social research discovering the gap between the academic research and the real industrial application, especially in the context of the energy issue. The main objectives of this social research aim at socially figuring out the developing background of the energy issue as well as getting prepared for the further industrialization of our project. We want to hear from different opinions from various aspects, including the public, the academic, the industrial, and also the authority. Only when concerns from all these parties are understood, could we fully comprehend the issue in real social context, and then figure out a series of feasible solutions to profoundly contribute to our society.<p></p>
 
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Our social research started as early as we just decided to work on the energy issue. We built up a long-term real-time interaction with the society, instead of a simple short glimpse, for the comprehensively understanding on the issue requires continuous communications and thoughts exchanging. Generally speaking, this social research consisted of two main parts, the public questionnaire and interviews. A public questionnaire was implemented to be familiar to the general public attitudes. We carefully analyzed the data collected. And according to the main concerns and opinions we found through the data analysis, we invited several experts from different walk of to participate in a series of theme interviews. Our interviewees included scientists working at the cutting-edge in the field, industrial representatives dedicating to the energy conservation, as well as from the administrative government. With their generous engagements, our team was able to further explore the project in a social context.<p></p>
 
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<a href="https://2016.igem.org/Team:ShanghaitechChina/HP/Silver#6">Click here to know more about our social research.</a>
 
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        <h1 align="center">Questionnaire</h1>
 
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A questionnaire on the energy issue was designed and then distributed through the internet. All 37 questions were asked, and here, we performed the core analysis of the questionnaire. We have finally in all collected 231 samples, 15 of which were canceled as invalid samples due to their short answering time (less than 180 seconds). The rest of 216 samples were regarded as valid responds that qualified for further analysis.
 
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<a href="https://2016.igem.org/Team:ShanghaitechChina/HP/Silver#7">Click here to view detailed questionnaire analysis.</a>
 
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<a href="https://2016.igem.org/Team:ShanghaitechChina/HP/Silver#8">Click here to find further information about interviews.</a>
 
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           <h1 align="center">Team Collaboration</h1>
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<a href="https://2016.igem.org/Team:ShanghaitechChina/Collaborations#9">Team Collaborations</a>
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<h3>>Contribution:</h3>
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<h4>
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<li>Biobrick: <a href="http://parts.igem.org/Part:BBa_K1583003">BBa_K1583003</a>
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<li> Group: ShanghaitechChina</li>
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<li> Author: Lechen Qian, Shijie Gu</li>
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<li> Summary: We created new way to characterize this biobrick which was originally designed and characterized by iGEM15_TU_Delft. We utilize NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) in our project to improve the characterization. We demonstrated the validity of the approach for measurement of biofilm composed by CsgA-His density of <i>E. coli</i> curli system and think highly of this characterization for its general application in other biofilm systems. Also, we utilized TEM to help us scrutinize the binding effect in microscopic world.</li>
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<h3>>Improvement:</h3>
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<h4>Quantum dots binding test</h4>
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<p>
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In order to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles, we apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 1h incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and thus secreted biofilm, and firmly attached with QDS and thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgA-Histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow. The picture was snapped by ChemiDoc MP,BioRad, false colored.</p>
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<img src="https://static.igem.org/mediawiki/parts/f/f2/Shanghaitechchina_Histag%2BQDs.png" width="40%">
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<b>Fig. 1</b>：Binding test between CsgA-his and Quantum dots. The image was snaped by ChemiDoc MP,BioRad, false colored.
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<h4>Comparison test of Quantum dots Binding between CsgA-his and CsgA</h4>
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In order to prove the effect of binding between CsgA-Histag mutant and inorganic nanoparticles is distinct, we apply same amount of suspended CdSeS/ZnS QDs solution followed by the same procedure mentioned above. After 1h incubation, we used PBS washing 2 times. The picture verify our postulation: On the left, CsgA-Histag mutant were induced and its biofilm bind with QDS. CsgA biofilm without Histag cannot bind with QDs thus its red fluorescence is much weaker. </p>
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<img src="https://static.igem.org/mediawiki/parts/8/8e/Shanghaitechchina_part_153.png" width="40%">
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<b>Fig. 2</b>：Comparison test of Quantum dots Binding between CsgA-his and CsgA.
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<h4>CdS nanorods Templating </h4>
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As for biofilm characterization, transmission electron microscopy is frequently to be used to visualize the nanofiber network. However, TEM is not very efficient to visualize soft matter due to the less dense of elections produced on soft matter even after negative staining. Amazingly, after incubation with CdS nanorods , the biofilm areas are densely templated by better conductive materials such as CdS nanorods  and we can easily confirm the expression of biofilm.</p>
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<b>Fig. 3</b>：Representative TEM images of biotemplated CdS nanorods on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with nanorods for 1h, nanomaterials are densely attached to biofilm.
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<h4>Details see ShanghaitechChina team's <a href="https://2016.igem.org/Team:ShanghaitechChina/Notebook#biofilm">protocol</a></h4>
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           <h1 align="center">ShanghaiTech iGEM Day for Public Interests (iDPI)</h1>
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           <h1 align="center">Optimize the codon</h1>
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Aiming at spreading the iGEM as well as dedicating to various  social engagements, ShanghaiTech iGEM Day for Public Interests (iDPI) was found in 2016, along with the establishment of the first ever iGEM team at ShanghaiTech University. The iDPI at ShanghaiTech is set up to encourage every iGEM team to devote themselves not only into scientific researches, but also social practices.<p></p>
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In the acronym name iDPI, “D” stands for dedication, “P” stands for passion, and “I” stands for inspiration. Our hope is that with the establishment of iDPI, every iGEMer at ShanghaiTech would bear a strong social awareness in minds, to yield profound contributions and achieve social values.<p></p>
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This year, the iDPI collaborated with Shanghai Coordinate International School, holding an iGEM Lab Open House Day on Zhangjiang campus.<p></p>
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<a href="https://2016.igem.org/Team:ShanghaitechChina/Collaborations#10">Click here for more details about iDPI.</a>
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<h3>>Contribution:</h3>
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<li>Biobrick: <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>
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<li> Group: ShanghaitechChina</li>
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<li> Author: Yifan Chen</li>
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<img src="https://static.igem.org/mediawiki/2016/c/cf/Stu_%289%29.jpg" style="width:100%;">
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<li> Summary: We optimized [FeFe] Hydrogenases originally from the bacterium <i>Clostridium acetobutylicum</i> (Original coding sequence: hydA, <a href="http://parts.igem.org/Part:BBa_K535002">BBa_K535002</a>, designed by: iGEM11_UNAM-Genomics_ Mexico. Optimized coding sequence: hydA with SpyTag and Histag <a href="http://parts.igem.org/Part:BBa_K2132005">BBa_K2132005</a>) to accept electrons and therefor enable catalytic production of hydrogen in our project. The optimized coding sequence would produce more protein, theoretically. And optimization also improved the activity of [FeFe] Hydrogenases according to the experiment that we did.</li>
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<h3>>Improvement:</h3>
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<h4>Codon usage bias adjustment</h4>
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<p>We analysed the Codon Adaptation Index (CAI) of the optimized coding sequence and the original one. And the distribution of codon usage frequency along the length of the gene sequence is increased from 0.33 to 0.97. A CAI of 1.0 is considered to be perfect in the desired expression organism, and a CAI of > 0.8 is regarded as good, in terms of high gene expression level.</p>
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<b>Fig. 4</b>：The distribution of codon usage frequency along the length of the gene sequence.
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<p>We also compared the Frequency of Optimal Codons (FOP). The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression organism. As we can see, the percentage of 91-100 increased largely, from 36 to 86, after the optimization.</p>
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<b>Fig. 5</b>：The percentage distribution of codons in computed codon quality groups.
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<h4>What's more, we removed repeat sequences to break the Stem-Loop structures, which impact ribosomal binding and stability of mRNA.</h4>
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      <th><strong>Max Direct Repeat</strong></th>
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      <th><strong>Max Inverted Repeat</strong></th>
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      <th><strong>Max Dyad Repeat</strong></th>
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      <td>After Optimization</a></td>
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      <td>Size:15 Distance:3 Frequency:2</td>
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      <td>None</td>
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      <td>None</td>
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      <td>Before Optimization</a></td>
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      <td>Size:16 Distance:231 Frequency:2</td>
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      <td>None</td>
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      <td>Size: 13 Tm: 34.6 Start Positions: 680, 1357</td>
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      <td colspan="12" align="center"><strong>Table 1: Removed repeat sequences information</strong></td>
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<h3>>Conclusion:</h3>
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<p>A wide variety of factors regulate and influence gene expression levels, and after taking into consideration as many of them as possible, OptimumGene™ produced the single gene that can reach the highest possible level of expression.</p>
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<p>In this case, the native gene employs tandem rare codons that can reduce the efficiency of translation or even disengage the translational machinery. We changed the codon usage bias in <em>E. coli</em> by upgrading the CAI from 0.33 to 0.97 . GC content and unfavorable peaks have been optimized to prolong the half-life of the mRNA. The Stem-Loop structures, which impact ribosomal binding and stability of mRNA, were broken.</p>
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<img src="https://static.igem.org/mediawiki/2016/e/ea/SHTU_D3.png" width="70%">
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<b>Fig. 6</b>：The protein alignment of new and old protein.
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Latest revision as of 22:56, 19 October 2016

igem2016:ShanghaiTech