Difference between revisions of "Team:ShanghaitechChina/Demonstrate"
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| − | <a href="# | + | <a href="#Overview">Overview</a> |
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| − | <a href="#Method and Instrument | + | <a href="#Method and Instrument">Method and Instrument</a> |
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| − | <a href="#AResults | + | <a href="#AResults">Results</a> |
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| − | <a href="#Further Exploration | + | <a href="#Further Exploration">Further Exploration</a> |
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| − | <a href="#Conclusion | + | <a href="#Conclusion">Conclusion</a> |
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| − | <a href="#p10 | + | <a href="#p10">Applied Design</a> |
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<img class="imgnav" src="https://static.igem.org/mediawiki/2016/9/9d/T--ShanghaitechChina--title-demonstration.png"> | <img class="imgnav" src="https://static.igem.org/mediawiki/2016/9/9d/T--ShanghaitechChina--title-demonstration.png"> | ||
| − | <p id=" | + | <p id="Overview"></p> |
<div class="content" > | <div class="content" > | ||
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<div class="col-lg-12" > | <div class="col-lg-12" > | ||
| − | <h1 align="center" > | + | <h1 align="center" >Overview</h1> |
<p></p> | <p></p> | ||
We aimed to design a biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that harnesses the energy of sun light. Biofilm-anchored nanorods can efficiently convert photons to electrons, which seamlessly tap into the electron chain of engineered strain carrying FeFe hydrogenase gene cluster, thereby achieving high-efficiency hydrogen production. It is noteworthy that our system facilitates the recycling of the expensive nanorods as the biofilms were grown on easy-separation micro-beads to anchored NRs. | We aimed to design a biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that harnesses the energy of sun light. Biofilm-anchored nanorods can efficiently convert photons to electrons, which seamlessly tap into the electron chain of engineered strain carrying FeFe hydrogenase gene cluster, thereby achieving high-efficiency hydrogen production. It is noteworthy that our system facilitates the recycling of the expensive nanorods as the biofilms were grown on easy-separation micro-beads to anchored NRs. | ||
<p></p> | <p></p> | ||
| − | The demonstration starts from the hydrogen production assay of the system made of all the components, biofilm anchored CdS on microspheres and the strain expressing FeFe hydrogenase. Notably, our hydrogen production has shown great efficiency compared to some precursors using hydrogenase. This section demonstrates the hydrogen production by integrating biofilm-anchored NRs with strain harboring hydrogenase gene clusters. For a thorough description of what we have achieved, please refer to | + | The demonstration starts from the hydrogen production assay of the system made of all the components, biofilm anchored CdS on microspheres and the strain expressing FeFe hydrogenase. Notably, our hydrogen production has shown great efficiency compared to some precursors using hydrogenase. This section demonstrates the hydrogen production by integrating biofilm-anchored NRs with strain harboring hydrogenase gene clusters. For a thorough description of what we have achieved, please refer to <a href="https://2016.igem.org/Team:ShanghaitechChina/IBS">Integrative Biohydrogen System</a>. <p></p> |
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| − | <p id=" | + | <p id="Method and Instrument"></p> |
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<div class="col-lg-12"> <center> <h1 > Method and Instrument</h1></center> | <div class="col-lg-12"> <center> <h1 > Method and Instrument</h1></center> | ||
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<h2> Method </h2> | <h2> Method </h2> | ||
| − | The biofilm, whose subunit was CsgA engineered with HisTag on N-termial and SpyCachter-HisTag on C-terminal, was grown on microspheres, 25 micrometers in diameter for 48 hours. NR’s (7.72*10 | + | The biofilm, whose subunit was CsgA engineered with HisTag on N-termial and SpyCachter-HisTag on C-terminal, was grown on microspheres, 25 micrometers in diameter for 48 hours. NR’s (7.72*10<sup>-9</sup> M) were then added and given 30 min to bind to the HisTag on CsgA subunit. (The engineered SpyCatcher was used for possible pure hydrogenase binding, our alternative proposal.) The solution was centrifuged and the sediments contained biofilm beads covered with NR. This sediment was resuspended in PBS and was added to the reaction solution consisting of <em>E. coli</em> with engineered hydrogenase (wet weight 100ug) resuspended in PBS, 150Mm NaCl, 100mM VitaminC, and mediator solution (5mM Paraquat dichloride, for mediating the electrons across the cell membrane). The whole solution including bacteria is adjusted to pH=4 by 100mM Tris-HCl(pH=7.0), given that the pH of 4 was reported to be an optimal environment. [*]<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature. |
| − | In the activity assay of the hydrogenase in producing hydrogen, the system goes through three periods of “light-on and light-off”. The result (see below) shows the stability of the system and the reversible catalytic activity of the hydrogenase of the reaction, 2H+ + 2e- | + | In the activity assay of the hydrogenase in producing hydrogen, the system goes through three periods of “light-on and light-off”. The result (see below) shows the stability of the system and the reversible catalytic activity of the hydrogenase of the reaction, 2H<sup>+</sup> + 2e<sup>-</sup> -> H<sub>2</sub> .<p></p> |
<h2> Instrument</h2> | <h2> Instrument</h2> | ||
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</div></div></div></div> | </div></div></div></div> | ||
<p id="Assay"></p> | <p id="Assay"></p> | ||
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<center> <h1 > Results</h1></center> | <center> <h1 > Results</h1></center> | ||
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<b> > Calculating the hydrogen evolution rate of our integrated system.</b><p></p> | <b> > Calculating the hydrogen evolution rate of our integrated system.</b><p></p> | ||
| − | We | + | We calculated the hydrogen production efficiency using the standard curve. Specifically, we chose the data from the first hydrogen production period. We converted the data in mV into umol/L. We compared the efficiency of our system with previous work ( See reference 1.) . |
<center><img src="https://static.igem.org/mediawiki/2016/3/30/T--ShanghaitechChina--biaozhuanqingqibiaodingquxian.png"></center> | <center><img src="https://static.igem.org/mediawiki/2016/3/30/T--ShanghaitechChina--biaozhuanqingqibiaodingquxian.png"></center> | ||
<p style="text-align:center"><b>Figure Standard</b> Relationship between voltage data and concentration.</p> | <p style="text-align:center"><b>Figure Standard</b> Relationship between voltage data and concentration.</p> | ||
| − | + | Following the method above , we obtain the rate of hydrogen evolution: the tip of the first period is 7.061 mV at 500s. This corresponds to 2.179 (0.3086*7.061) umol/L at 500s. Thus the rate is 0.0126 (2.179/500*3mL*1000) umol/s, for 0.1g <em>E.coli</em>. In comparison with the rate from reference 1, 0.0086mol umol/s. This 46% increase in the efficiency shows that our system not only functions, but is also a big improvement compared with a artificial hydrogen production system reported before .<p></p> | |
</div></div></div> | </div></div></div> | ||
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<div class="col-lg-12" > | <div class="col-lg-12" > | ||
<h1 align="center" >Conclusion</h1> | <h1 align="center" >Conclusion</h1> | ||
| − | In conclusion, E. | + | In conclusion, <em>E.coli</em> strains expressing biofilm on microspheres to anchor nanorods and strains expressing hydrogenase work great together for producing hydrogen. Although the system is not as efficient as the one with nanorods flowing freely due to some possible reasons as the size of microsphere, the system still achieved a fairly good hydrogen production rate compared to the similar precursor reported this year. Since the use of microsphere allowed easy recycling of the expensive nanomaterials, we therefore propose this model as our final model, although further optimization of the system is still under way, including deciding on the optimized material and size the microsphere for biofilm growth. Meanwhile, our SpyCatcher on the CsgA allows the binding of other proteins that may significantly improve our system. This will lead to our future work. Stay tuned, or you may want to join us in this project as well: Contact us: zhongchao@shanghaitech.edu.cn Investor are also welcome.<p></p> |
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<p> Given that our system has been proved to work with repeatability and its intrinsic characteristics for massive application, we think our project is a successful applied design. Here, we summarize the qualification and merits of our design in the following figure. </p> | <p> Given that our system has been proved to work with repeatability and its intrinsic characteristics for massive application, we think our project is a successful applied design. Here, we summarize the qualification and merits of our design in the following figure. </p> | ||
<center> | <center> | ||
| − | <img src=" https://static.igem.org/mediawiki/2016/ | + | <img src="https://static.igem.org/mediawiki/2016/0/09/T--ShanghaitechChina--hahahaNEW.jpg" width="80%"> |
</center> | </center> | ||
<p></p><p></p> | <p></p><p></p> | ||
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<p>[*] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H_2 Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.</p> | <p>[*] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H_2 Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.</p> | ||
| − | [1] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H2, Production of a Whole-Cell Reaction by Recombinant Escherichia coli, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016 | + | [1] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H2, Production of a Whole-Cell Reaction by Recombinant <em>Escherichia coli</em>, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016 |
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Latest revision as of 22:49, 19 October 2016
