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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  <h1>Solar Hunter System</h1>
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The recent work based upon Bacterial (M. thermoacetica)-quantum dots hybrid system to harvest value-based products has suggested great future for artificial photosynthesis system [1]. Despite important advances, the current efficiency and scope of application have been limited due to the damage of quantum dots on biological systems arising from direct contact of quantum dots with the cell membrane, the less efficient integration between bio-abiotic interfaces as well as the poor conductivity of most biological systems. To address these issues, we develop a solar hunter platform that can seamlessly integrate conductive bacteria biofilms, the high-efficiency photon-electron transformation of quantum dots with an efficient metabolic pathway of biological systems.  
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          <h1 align="center">Overview</h1>
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This year, we have done the two improvement work:<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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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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          <h1 align="center">Improve the characterization</h1>
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<h3>>Contribution:</h3>
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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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The biofilm system that came into our sight is type IV pili in Geobacter sulfurreducens, which is conductive microbial nanowire [2].  The wire can be expressed in genetically manipulated strains as long wires with binding sites for quantum dots and efficiently conduct electrons.  With the more surface area of biofilms for quantum dots and indirect contact between quantum dots with cell membrane, we expect a significant boost in the energy of light harvested by our Solar Hunter without sacrificing normal cell growth and regeneration.
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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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Specifically, we propose three demo examples here based on our newly developed artificial photosynthesis.  The first one is based on non-conductive biofilm, as for second and third one, we use conductive biofilm, which will be an increasingly complicated  system.
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1) At first, we want to establish the Solar Hunter system on E.Coli, whose biofilm serves as a synthetic nonconductive biological platform for self-assembling function materials. The amyloid protein CsgA , which is the dominant component in E.Coli, can be programmed to append small peptide domain and successfully secreted with biological functions. Then we propose that our Hunter family member can be an enzyme.  Nitrogenase complex is the central enzyme in the natural nitrogen-fixing process.  Previous researches have demonstrated the viability of using semiconductor CdS nanorod to harvest light and supply the electrons as a substitute for the Fe protein in the complex where electrons are generated from ATP [3].  The heterotetrameric MoFe protein, the other part in nitrogenase complex, will use the electrons provided to reduce N2 to NH3.  We will explore the possibility of an increase in the efficiency of the semiconductor-enzyme system usingE.Coli’s biofilm, on which biofilm subunit are engineered with SpyTag and SpyCatcher system from FbaB protein to provide binding sites for proteins [4].
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2) 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 [5].  Geobacter can naturally express nanowires and transfer electrons between each other or do direct interspecies electron transfer(DIET) with other microorganisms; for our project, Methanosarcina.  Extern light is absorbed by the Geobacter and is transferred into electrons.  Semi-conductors are bind to the biofilm of Geobacter to enhance its conductivity. The electrons are then transported to Methanosarcina in the form of succinate and fumarate, used as the input material to produce value-added products like methane.
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3) In addition, solar hunters will include a pathway for leucine synthesis from acetate (acetyl-coenzyme A) [6], since leucine is of higher value.  They use carbon dioxide as a carbon source to synthesize isoleucine via a combination of two pathways.  The first pathway is the acetyl-coenzyme A (acetyl-CoA) pathway [7], gaining electrons to reduce carbon dioxide and synthesizing acetyl-CoA which is a vital intermediate.  As acetyl-CoA is synthesized, it can be the raw material of the second pathway, which is the pathway for isoleucine biosynthesis in G. sulfurreducens, to give the final product isoleucine [8].  There are three main reasons for us to choose this combination.  Firstly, these two pathways are found in G. sulfurreducens.  Secondly, carbon dioxide is a kind of environmentally friendly carbon source.  Thirdly, comparing to carbon dioxide, isoleucine is a high value-added chemical that will bring us a high level of economic efficiency.  Additionally, the second pathway can be replaced by other pathways to synthesize other value-added chemicals, such as butanol.
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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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Collectively, we envision that these three parallel systems should build a powerful solar Hunter system to push the boundary of current artificial photosynthesis.  
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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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  [1] Sakimoto K K, Wong A B, Yang P. Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production.[J]. Science, 2016, 351(6268):74-77.
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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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[2] Strycharz S M, Glaven R H, Coppi M V, et al. Gene expression and deletion analysis of mechanisms for electron transfer from electrodes to Geobacter sulfurreducens[J]. Bioelectrochemistry, 2011, 80(2):142-150.
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<img src="https://static.igem.org/mediawiki/parts/e/e1/Shanghaitechchina_CsgAHistag%2Bnanorods.png" width="90%">
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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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[3] Brown K A, Harris D F, Wilker M B, et al. Light-driven dinitrogen reduction catalyzed by a CdS:nitrogenase MoFe protein biohybrid.[J]. Science, 2016, 352(6284):448-450.
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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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[4] Zakeri B, Fierer J O, Celik E, et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(12):690-7.</p>
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[5] Rotaru A E, Shrestha P M, Liu F, et al. A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane[J]. Journal of Virology, 2013, 18(1):324-31.</p>
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[6] Risso C, Van Dien S J A, Lovley D R. Elucidation of an Alternate Isoleucine Biosynthesis Pathway in Geobacter sulfurreducens[J]. Infection Control & Hospital Epidemiology, 2008, 190(7):277-81.</p>
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[7] Methé B A, Nelson K E, Eisen J A, et al. Genome of Geobacter sulfurreducens: metal reduction in subsurface environments.[J]. Science, 2003, 302(5652):1967-9.</p>
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          <h1 align="center">Optimize the codon</h1>
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[8] Mahadevan R, Palsson B Ø, Lovley D R. In situ to in silico and back: elucidating the physiology and ecology of Geobacter spp. using genome-scale modelling.[J]. Nature Reviews Microbiology, 2011, 9(1):39-50.</p>
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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>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