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+ | <a href="#AResults" style="font-size:14px;">Results</a> | ||
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− | <a href="# | + | <a href="#Potential use for wider application">Potential use for wider application</a> |
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<img class="imgnav" src="https://static.igem.org/mediawiki/2016/0/01/T--ShanghaitechChina--member--bf--Integrative_Biohydrogen_System.png"> | <img class="imgnav" src="https://static.igem.org/mediawiki/2016/0/01/T--ShanghaitechChina--member--bf--Integrative_Biohydrogen_System.png"> | ||
− | < | + | <p id="Abstract" ></p> |
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<h1 align="center" style="font-weight:bold">Abstract</h1> | <h1 align="center" style="font-weight:bold">Abstract</h1> | ||
− | <p>Artificial photosynthesis represents a promising solution for energy issues, however, the efficiency, robustness, and scalability does not meet the requirements of industrial applications. We proposed and demonstrated a sun-powered biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that could potentially solve the issues above. 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. Furthermore, 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 some precursors using hydrogenase. Practically speaking, the system comprising E. Coli 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. The efficiency, recyclability, stability, scalability, and versatility makes our system a design that is truly applicable.</p> | + | <p><strong>Artificial photosynthesis represents a promising solution for energy issues, however, the efficiency, robustness, and scalability does not meet the requirements of industrial applications.</strong> We proposed and demonstrated a sun-powered biofilm-interfaced artificial hydrogen-producing system, Solar Hunter, that could potentially solve the issues above. 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. Furthermore, 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 some precursors using hydrogenase. Practically speaking, the system comprising <em>E. Coli</em> 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.<strong> The efficiency, recyclability, stability, scalability, and versatility makes our system a design that is truly applicable.</strong></p> |
<center><img src="https://static.igem.org/mediawiki/2016/5/5d/Plan_1_V2.jpg" style="width:52%"></center> | <center><img src="https://static.igem.org/mediawiki/2016/5/5d/Plan_1_V2.jpg" style="width:52%"></center> | ||
</div></div></div> | </div></div></div> | ||
+ | <p id="Assay" ></p> | ||
<div id="Assay" class="content"> | <div id="Assay" class="content"> | ||
− | <div class="row"> | + | <div class="row"><p id="APrinciple"></p> |
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<h2 align="center" style="font-weight:bold">Hydrogenases Expression and Enzyme Activity Assay</h2> | <h2 align="center" style="font-weight:bold">Hydrogenases Expression and Enzyme Activity Assay</h2> | ||
− | <h3 | + | <h3>(1) Principles and Methods</h3> |
− | In the activity assay of the hydrogenase in producing hydrogen, we repeated three parallel experiments to test the activity and validated the repeatability of our rudimentary system. In each parallel experiment, the system goes through three periods of “light-on and light-off”. The results (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, we repeated three parallel experiments to test the activity and validated the repeatability of our rudimentary system. In each parallel experiment, the system goes through three periods of “light-on and light-off”. The results (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> |
The three parallel systems consist of <em>E. coli</em> with engineered hydrogenase (wet weight 100ug) resuspended in PBS, 200ul quantum dots/nanorods (7.72*10^-9 M) 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. The whole system is based on former study. <span style=”font-size:12px”></span><p></p> | The three parallel systems consist of <em>E. coli</em> with engineered hydrogenase (wet weight 100ug) resuspended in PBS, 200ul quantum dots/nanorods (7.72*10^-9 M) 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. The whole system is based on former study. <span style=”font-size:12px”></span><p></p> | ||
In addition, we did a fourth assay with resuspended microspheres covered with quantum dots/nanorods bound biofilm in PBS, in place of the resuspended quantum dots/nanorods solution. The fourth set is the actual system we are proposing, since it is as efficient and allows the recycling of quantum dots/nanorods.<p></p> | In addition, we did a fourth assay with resuspended microspheres covered with quantum dots/nanorods bound biofilm in PBS, in place of the resuspended quantum dots/nanorods solution. The fourth set is the actual system we are proposing, since it is as efficient and allows the recycling of quantum dots/nanorods.<p></p> | ||
In our experiment, we find that despite the reported affected catalytic ability of FeFe hydrogenase due to oxygen, non-strict anaerobic and short-term exposure to oxygen does not cause detrimental effects on the enzyme activity of producing hydrogen. This can be explained by the high catalytic ability and the segregation layer from the atmosphere provided by the hydrogen it produces. Meanwhile, the electron sacrificial agent VitaminC also adds to the “protection layer” of the hydrogenase in our system.<p></p> | In our experiment, we find that despite the reported affected catalytic ability of FeFe hydrogenase due to oxygen, non-strict anaerobic and short-term exposure to oxygen does not cause detrimental effects on the enzyme activity of producing hydrogen. This can be explained by the high catalytic ability and the segregation layer from the atmosphere provided by the hydrogen it produces. Meanwhile, the electron sacrificial agent VitaminC also adds to the “protection layer” of the hydrogenase in our system.<p></p> | ||
− | < | + | <p id="AInstrument" style="margin-bottom:80px"></p> |
+ | <h3>(2) Instrument</h3> | ||
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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)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); (2) an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a date 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)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); (2) an anaerobic reaction container which is a transparent circular cuvette that allows light to go through; (3) a hydrogen electrode linked to its inner sensor inserted into the reaction container to measure the realtime concentration of hydrogen; (4) a date 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> | ||
+ | <p id="AResults" style="margin-bottom:80px"></p> | ||
+ | <h3 >(3) Results</h3> | ||
<h4><b>a) Contribution of each component of the hydrogen production system</b></h4> | <h4><b>a) Contribution of each component of the hydrogen production system</b></h4> | ||
The first hydrogen production data using our system is the pink curve (curve 1) in Figure 1. It shows that lighting can induce hydrogen production in a closed system with nano rods (NR), mediator Methyl Viologen, and IPTG-induced bacteria transformed with fused plasmid. To prove that every element of the system is necessary and that it is our hydrogenase that produced the hydrogen rather than NR, we conducted a series of experiments.<p></p> | The first hydrogen production data using our system is the pink curve (curve 1) in Figure 1. It shows that lighting can induce hydrogen production in a closed system with nano rods (NR), mediator Methyl Viologen, and IPTG-induced bacteria transformed with fused plasmid. To prove that every element of the system is necessary and that it is our hydrogenase that produced the hydrogen rather than NR, we conducted a series of experiments.<p></p> | ||
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Another step in proving that it is that the hydrogenase is indeed responsible for hydrogen production is to contrast the production level between the induced and un-induced bacteria suspension. The experiment we conducted are summarized in Figure 6B In this set of experiment, the blue line (curve 4) acts as our blank control. In this group, we use the wild type BL21 cells without plasmid. Although we can see a positive oscillation during a short time in the curve, the production was not at high rate and is likely due to the native hydrogenase in <em>E. coli</em>. The green curve (curve 5) represents the transformed bacterial with no induction of IPTG after 12h cultivation. The flat curve shows that it could not produce hydrogen, which proves that the induction of the hydrogenase expression is necessary. To further confirm, we did another experiment using bacteria that have grown 36 hours with no induction. The purple curve (curve 6) clearly contrasts the induced BL21 and the non-induced one. With curve 4 to 6, we have demonstrated that, with the help of NR, it was our hydrogenase in the system that produced the hydrogen we detected.<p></p> | Another step in proving that it is that the hydrogenase is indeed responsible for hydrogen production is to contrast the production level between the induced and un-induced bacteria suspension. The experiment we conducted are summarized in Figure 6B In this set of experiment, the blue line (curve 4) acts as our blank control. In this group, we use the wild type BL21 cells without plasmid. Although we can see a positive oscillation during a short time in the curve, the production was not at high rate and is likely due to the native hydrogenase in <em>E. coli</em>. The green curve (curve 5) represents the transformed bacterial with no induction of IPTG after 12h cultivation. The flat curve shows that it could not produce hydrogen, which proves that the induction of the hydrogenase expression is necessary. To further confirm, we did another experiment using bacteria that have grown 36 hours with no induction. The purple curve (curve 6) clearly contrasts the induced BL21 and the non-induced one. With curve 4 to 6, we have demonstrated that, with the help of NR, it was our hydrogenase in the system that produced the hydrogen we detected.<p></p> | ||
<h4><b>b) Bidirectional catalytic property of [FeFe] hydrogenase</b></h4> | <h4><b>b) Bidirectional catalytic property of [FeFe] hydrogenase</b></h4> | ||
− | As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen ( | + | As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen (<sub>2</sub>). Thus, when we “turn off” the production mode, we should be able to see the consumption of hydrogen by hydrogenase. In testing this bidirectional catalytic property, conducted an experiment where we turned on and turned off the light alternately. The data is shown below in Figure 3. During lighting period, the hydrogen production increases, until we shut off the light at points that correspond to the tips. The curve then goes downward, showing that the hydrogen concentration is lowered, an evidence of the consumption of hydrogen. It is noteworthy that the hydrogenase shows the greatest production rate at the beginning of lighting: a transient sharp rise can be observed at the valleys. It is also obvious that each period of “light-on light-off” gives similar curves, which implies that our hydrogenase is stable. |
<center><img src="https://static.igem.org/mediawiki/2016/a/ab/T--ShanghaitechChina--asasy--bidirectlycat.png"></center> | <center><img src="https://static.igem.org/mediawiki/2016/a/ab/T--ShanghaitechChina--asasy--bidirectlycat.png"></center> | ||
<p style="text-align:center"><b>Figure 3</b> Verifying the bidirectional catalytic property of [FeFe] hydrogenase.</p> | <p style="text-align:center"><b>Figure 3</b> Verifying the bidirectional catalytic property of [FeFe] hydrogenase.</p> | ||
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<p style="text-align:center"><b>Figure5.</b> Repeatability of our hydrogen production system. The three curves conform. This demonstrates our preliminary prototype as repeatable and robust. </p> | <p style="text-align:center"><b>Figure5.</b> Repeatability of our hydrogen production system. The three curves conform. This demonstrates our preliminary prototype as repeatable and robust. </p> | ||
− | To further prove our system as a reliable one, we did three sets of hydrogen production assays in one day in a row (Figure5, curve 10-12). The system was mainly made up of resuspended CdS nonorods, E. Coli BL21 transformed with the plasmid containing all the four [FeFe]hydrogenase subunits from Clostridium. acetobutylicum, and mediator, methyl viologen. (See Figure1 or the section of Principles and Methods for details of the reaction system.) From the data shown, we clearly see the conformation of the three curves. This demonstrates our preliminary prototype in Figure1 as repeatable and robust. | + | To further prove our system as a reliable one, we did three sets of hydrogen production assays in one day in a row (Figure5, curve 10-12). The system was mainly made up of resuspended CdS nonorods, <em>E. Coli</em> BL21 transformed with the plasmid containing all the four [FeFe]hydrogenase subunits from Clostridium. acetobutylicum, and mediator, methyl viologen. (See Figure1 or the section of Principles and Methods for details of the reaction system.) From the data shown, we clearly see the conformation of the three curves. This demonstrates our preliminary prototype in Figure1 as repeatable and robust. |
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+ | <p id="Potential use for wider application"></p> | ||
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+ | <div class="col-lg-12" > | ||
+ | <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> | ||
+ | <div class="col-lg-12"> | ||
+ | <h3 class="bg" >SpyTag and SpyCatcher [3]</h3> | ||
+ | <h4><b>Introduction and Motivation: SpySystem</b></h4> | ||
+ | We want to attach enzymes to biofilm, so we turn to a widely applied linkage system, SpyTag and SpyCatcher.<p></p> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/c/c5/Shanghaitechchina_spy1.png" style="width:50%;"> | ||
+ | </center> | ||
+ | <h4><b> Design</b></h4> | ||
+ | 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> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/0/07/Shanghaitechchina_spy2.png" style="width:50%;"> | ||
+ | </center> | ||
+ | </div> | ||
+ | <div> | ||
+ | |||
+ | <h4><b>Characterization</b></h4> | ||
+ | As Figure6 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> | ||
+ | <center> | ||
+ | <img src="https://static.igem.org/mediawiki/parts/5/5c/Shanghaitechchina_mcherry-SpyTag%2BCsgA-SpyCatcher.png" style="width:100%;"> | ||
+ | </center> | ||
+ | <p style="text-align:center"><b>Fig 6. </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> | ||
+ | </div> | ||
+ | |||
+ | </div></div></div> | ||
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+ | |||
+ | <p id="Reference"></p> | ||
+ | <div class="content"> | ||
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− | <p>[1] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and | + | <p>[1] Cao Y, Bai X F. Progress in Research of Preparation of Loaded Nano-CdS and H<sub>2</sub> Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.</p> |
− | <p>[2] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic | + | <p>[2] Honda Y, Hagiwara H, Ida S, et al. Application to Photocatalytic H<sub>2</sub>, Production of a Whole-Cell Reaction by Recombinant <em>Escherichia coli</em>, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016</p> |
+ | <p>[3] Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).</p> | ||
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Latest revision as of 02:10, 20 October 2016