Difference between revisions of "Team:ShanghaitechChina/Description"

 
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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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   Description
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  The combination of artificial and biological photosynthesis has shown great efficiency in recent work with M. thermoacetica-CdS hybrid system to produce acetic acid (acetyl-coenzyme A) [1]. Our project is inspired by this solar-to-chemical synthesis and we call our project as Solar Hunter. Rather than using biologically precipitated CdS nanoparticals, Hunter will exploit the proteins on the biofilm to bind CdS or other compatible quantum dots. The protein that came into our sight is pili, the microbial nanowire[2]. The wire can be expressed in genetically manipulated strains as long wires with binding sites for quantum dots. With the more space made for more quantum dots, we expect a boost in the energy of light harvested by our Solar Hunter. In addition, Hunter will include a pathway for leucine synthesis from acetate (acetyl-coenzyme A)[3], since leucine is of higher value.
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  The solar source in the solar-chemical system is, in its essence, energy with electrons. In an attempt to apply our quantum dots-pili hybrid to a wider extent, we decide to try out this model on another amazing archaea, Methanosaeta barundinacea, which is likely to have a pathway to simply use carbon dioxide, electrons and protons for the biosynthesis of methane[4].
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          <h1 align="center">Overview</h1>
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  Our Hunter family member can be just a protein as well. Nitrogenase complex is the central enzyme in the natural nitrogen-fixing process. Previous researches have demonstrated the viability of the using semiconductor-protein hybrid  to harvest electrons from sunlight as a substitute for the Fe protein in the complex where electrons are generated from ATP. [5] Aiming to construct a well-established nitrogen fixation platform, we will explore the possibility of an increase in the efficiency of the system using Hunter’s pili, mediated by Spytag and Spycatcher system.
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This year, we have done the two improvement work:<p></p>
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  These three parallel systems should altogether build a powerful Hunter family to bring the endless energy from the sun for the good of mankind.
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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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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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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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  [1] 10.1126/science.aad3317
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  [2] 10.1016/j.bioelechem.2010.07.005
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          <h1 align="center">Improve the characterization</h1>
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  [3] 10.1128/JB.01841-07
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  [4] 10.1039/Energy Environ.Sci.c3ee42189a
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<h3>>Contribution:</h3>
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  [5] 10.1126/science.aaf2091
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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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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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<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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<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">Optimize the codon</h1>
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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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<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> </strong></th>
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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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<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