<a href="#p10">Overview</a>

<a href="#Introduction to Solar Hunter">Introduction to Solar Hunter</a>

<a href="#Demonstrated Functionality" >Demonstrated Functionality</a>

<a href="#Methods"style="font-size:14px" >Methods</a>

<a href="#Instrument" style="font-size:14px">Instrument</a>

<a href="#Results"style="font-size:14px" >Results</a>

</ul>

<a href="#Efficiency">Efficiency</a>

<a href="#Potential use for wider application" >Potential use for wider application</a>

<a href="#Conclusion" >Conclusion</a>

         <h1 align="center">Overview</h1>

<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>

<img src="https://static.igem.org/mediawiki/2016/0/09/T--ShanghaitechChina--hahahaNEW.jpg" width="80%">

<p id="Introduction to Solar Hunter"></p>

<p id="Demonstrated Functionality"></p>

<p id="Methods"></p>   

     <div class="col-lg-12">      <center> <h1 >Demonstrated Functionality</h1></center>

<h2> Methods </h2>

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>

<h2> Results </h2>

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.) .

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>

         <h1 align="center"  >Potential use for wider applications</h1>

<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>

               <h3 class="bg" >SpyTag and SpyCatcher [2]</h3>

We want to attach enzymes to biofilm, so we turn to a widely applied linkage system, SpyTag and SpyCatcher.<p></p>

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>

<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>

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.

[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>