Difference between revisions of "Team:ShanghaitechChina/Design"

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         <h1 align="center"> Applied Design</h1>
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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>
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<p> Given that our integrative system has been proved to produce hydrogen with great stability and repeatability and its intrinsic characteristics for scalable production, we think our project is a successful applied design. Here, we summarize the qualification and merits of our design in the following figure.  In addition, biofilm-interfaced Nanorods has the potential for easy recycling and is cheap for production, thus presenting an innovative and interesting example towards industrial-oriented artificial photosynthesis system. </p>
 
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<img src=" https://static.igem.org/mediawiki/2016/1/10/T--ShanghaitechChina--AP.pdf" width="80%">
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     <div class="col-lg-12">      <center> <h1 > Method and Instrument</h1></center>
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     <div class="col-lg-12">      <center> <h1 >Demonstrated Functionality</h1></center>
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<h2> Method </h2>
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<h2> Methods </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^-9 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. [1]<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature.  
 
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^-9 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. [1]<span style=”font-size:12px”> </span> Prior to the assay, the <em>E. coli</em> was induced with IPTG overnight at room temperature.  
  
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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-  ⇿ H2 .<p></p>
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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-  ⇿ H2 .<p></p>
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<center><p style="text-align:center"><b>Figure 1</b> Apparatus of the hydrogen production assay.</p></center>
 
<center><p style="text-align:center"><b>Figure 1</b> Apparatus of the hydrogen production assay.</p></center>
 
It contains (1) an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (2) a light source in our hydrogen production assay acting as a substitute for the real sun. (We chose a high-power white LED light, set 28cm away from the reaction container for a even distribution of photons); (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a data hub; (5) a computer connected to the hub to record the data and generate the curve of concentration variation within a period of time. <p></p>
 
It contains (1) an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (2) a light source in our hydrogen production assay acting as a substitute for the real sun. (We chose a high-power white LED light, set 28cm away from the reaction container for a even distribution of photons); (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a data hub; (5) a computer connected to the hub to record the data and generate the curve of concentration variation within a period of time. <p></p>
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<center><img src="https://static.igem.org/mediawiki/2016/4/4f/T--ShanghaitechChina--asasy-withfinalplan-bidirectlycat.png"></center>
 
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         <p style="text-align:center"><b>Figure 2</b> Hydrogen evolution curve with nanorods bound to biofilm beads.</p>
 
         <p style="text-align:center"><b>Figure 2</b> Hydrogen evolution curve with nanorods bound to biofilm beads.</p>
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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>
  
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We are particularly interested in learning what our efficiency is compared to one study reported this year. See reference 1. In calculating the efficiency, we chose the data from the first hydrogen production period. We converted the data in mV into umol/L. The standard curve is provided by the lab who supervised our assay apparatus.  
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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>
 
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         <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>
  
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Thus, 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 E. Coli. In comparison with the rate from reference 1, 0.0086mol umol/s. This 46% increase in the efficiency shows that our system not only works, but is also a progress for the study of artificial hydrogen production system.<p></p>
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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 <i>E. coli</i>. 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>
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        <h1 align="center"  >Potential use for wider applications</h1>
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<b> Our biofilm system included a SpyCatcher system which has the potential for working directly with enzymes on biofilm. This design was not utilized in our hydrogen production so far, but it offers a gate for enzymes to directly react with nanomaterials since the CsgA subunit is engineered with both the Histag and SpyCatcher. This potential use might lead to a boost in efficiency in some nanomaterial-enzymes combinations. The reasoning of the design, and the proof of the functional design is shown below.  </b><p></p>
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              <h3 class="bg" >SpyTag and SpyCatcher [2]</h3>
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<h4><b>Introduction and Motivation: SpySystem</b></h4>
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We want to attach enzymes to biofilm, so we turn to a widely applied linkage system, SpyTag and SpyCatcher.<p></p>
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<h4><b> Design</b></h4>
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Appending SpyTag to CsgA subunit is a traditional and hackneyed approach to modify biofilm posttranscriptionally. Here, we challenge to attach larger part, SpyCatcher, to CsgA to enrich the versatility of biofilm platform. For one thing, we intend to pioneer new approach. For another aspect is that we concern SpyCatcher is too large that might jeopardize the biological activity and function of the enzyme. After comprehensive consideration, we decide to append SpyTag and SpyCatcher respectively to CsgA subunits and enzyme, and successfully prove their feasibility and stability.<p></p>
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<h4><b>Characterization</b></h4>
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As Figure3 illustrated, His-CsgA-SpyCatcher-Histag mutant incubated with mCherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicating the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–Histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on <i>E. coli</i>. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system. <p></p>
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<img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;">
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<p style="text-align:center"><b>Fig 3. </b> The first figures of each sample are snapped under green laser of 558 nm wavelength and mCherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria.. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.</p>
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         <h1 align="center"  >Conclusion</h1>
 
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In conclusion, E. Coli 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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In conclusion, <i>E. coli</i> strains expressing biofilm on microspheres to anchor nanorods and strains expressing hydrogenase work great together for producing hydrogen. The system achieved a fairly good hydrogen production rate compared to a work reported this year, a nearly 50% increase. The intrinsic adherence of biofilms towards various interfaces allows us to grow biofilms on easy-separation micro-beads, therefore facilitating recyclable usage of the biofilm-anchored NRs and endowing this whole system with recyclability. Notably, our hydrogen production has shown great stability compared to previous reports using hydrogenase. Practically speaking, the system comprising <i>E. coli</i> and biofilms are both amenable for scalable operation, rendering itself a great potential for large-scale industrial applications. Such system can also be adapted to other energy-oriented applications by utilizing engineered new strains with a diverse spectrum of enzymes or metabolic pathways. In addition, our SpyCatcher on the CsgA allows the binding of other proteins that may significantly improve our system. The demonstrated efficiency and stability, along with great potential in scalability, recyclability and versatility makes our system an innovative engineering design with potential for industrial application.
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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>   
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[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
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[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<p></p>
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[2] Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto <i>E. coli</i> curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).<p></p>
 
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Latest revision as of 23:09, 19 October 2016

igem2016:ShanghaiTech