Difference between revisions of "Team:ShanghaitechChina/IBS"

Latest revision as of 02:10, 20 October 2016

igem2016:ShanghaiTech

Hydrogenases Expression and Enzyme Activity Assay

(1) Principles and Methods

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^- ⇿ H₂ .

The three parallel systems consist of E. coli 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. Prior to the assay, the E. coli was induced with IPTG overnight at room temperature. The whole system is based on former study.

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.

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.

(2) Instrument

Figure 1 Apparatus of the hydrogen production assay.

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.

(3) Results

a) Contribution of each component of the hydrogen production system

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.

To see whether NR is necessary and whether the hydrogen is produced by the reaction between NR and water under lighting rather than our hydrogenase, we conducted the experiment where the system does not contain nano rods or contain only nano rods. The data is summarized in Figure 1A. The red curve (curve 2) represents the system with the transformed bacterial suspension but without nano rods (NR). The flat curve shows that the system without NR could not produce hydrogen with light; NR is necessary for the system. The black curve (curve 3) represents a system in which only NR and mediators are present, with no bacteria. The flat curve shows that it could not produce hydrogen, which proves that the elements of the bacteria is necessary in the synthesis of hydrogen.

Figure 2

click to enlarge the figure

Hydrogen production evolution curve (Sensor Data/ Hydrogen amount vs Time) with different components. The pink curve (curve 1) in all pictures is the hydrogen production with all the components, nano rods (NR), IPTG induction, and the bacteria transformed with our hydrogenase plasmid. The rest are data with one or two components missing. In particular, data in the integrated picture are categorized into Figure 2A and 2B. Figure 2A shows the system with or without nano rods or with nano rods alone, and Figure 2B represents the system with or without induction. The curve 3 in each of the specific figure is the blank control with not transformed E. coli BL21. This series of experiments show that only when both nano rods (NR) and IPTG-induced transformed bacteria are present can the system produce hydrogen in a stable way.

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 E. coli. 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.

b) Bidirectional catalytic property of [FeFe] hydrogenase

As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen (₂). 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.

Figure 3 Verifying the bidirectional catalytic property of [FeFe] hydrogenase.

During the period under lighting, 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.

c) Hydrogen production with nano rods suspension replaced by nano rods bound to biofilm beads.

Given the difficulty in recycling the nano rods due to their small size, we utilize biofilm to immobilize nano rods and aggregate them into larger assemblies that allow filtration or other ways of recycling including centrifugation. However, testing whether the NR aggregate work in our system is needed. We conducted experiments with nano rods suspension replaced by nano rods bound to biofilm beads. 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 were then added and given 30 min to bind to the HisTag on CsgA subunit. (The engineered SpyCatcher was used for future pure hydrogenase binding.) 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 system. The data is in Figure 3 In this experiment, we did the same “light-on light off” actions to the system and the pattern is similar to the one with NR suspension (Figure 2) During lighting, the rapid production of hydrogen can be clearly observed. Some other characteristics pertain, such as the sharp rise at the beginning of lighting.

Figure 4 Hydrogen production with nano rods suspension replaced by nano rods bound to biofilm beads.

We replaced the nanorods suspension with nano rods bound to biofilm beads. During the period with lighting, 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, as in Figure 3.

Comparing Figure 2 and Figure 3

In the process of hydrogen generation, a stir bar with a necessary speed of 800 RPM was used to generate the curve in Figure 3. But in Figure 4, a stir bar was not used. It is likely because the aggregates of NR have a bigger chance in colliding with E. coli to transfer electrons. We therefore propose this model as our final model, although further optimization of the system is still under way.

d) Repeatability

Figure5. Repeatability of our hydrogen production system. The three curves conform. This demonstrates our preliminary prototype 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, 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.

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+<li >
+<a href="#Abstract">Abstract</a>
+</li>
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 <a href="#Assay">Assay</a>
+<ul>
+<li>
+<a href="#APrinciple" style="font-size:14px;">Principle</a>
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-<a href="#APrinciple" style="font-size:14px;margin-left:15px;">Principle</a>
+<a href="#AInstrument" style="font-size:14px;">Instrument</a>
+</li>
+<li>
+<a href="#AResults" style="font-size:14px;">Results</a>
+</li>
+</ul>
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-<a href="#AInstrument" style="font-size:14px;margin-left:15px;">Instrument</a>
+<a href="#Potential use for wider application">Potential use for wider application</a>
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-<a href="#AResults" style="font-size:14px;margin-left:15px;">Results</a>
+<a href="#Reference">Reference</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">
+<p  id="Abstract" ></p>
+<div class="content">
    <div class="row">
+    <div class="col-lg-12">
+        <h1 align="center" style="font-weight:bold">Abstract</h1>
+<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>
+</div></div></div>
+<p  id="Assay" ></p>
+<div id="Assay" class="content">
+  <div class="row"><p  id="APrinciple"></p>
      <div class="col-lg-12">
           <h2 align="center" style="font-weight:bold">Hydrogenases Expression and Enzyme Activity Assay</h2>
-          <h3 id="APrinciple">(1) Principles and Methods</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-  ⇿ H2 .<p></p>
+          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>
 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>
-<h3 id="AInstrument">(2) Instrument</h3>
+<p  id="AInstrument" style="margin-bottom:80px"></p>
+<h3>(2) Instrument</h3>
+</div>
+  <div class="col-lg-6">
+<center><img src="https://static.igem.org/mediawiki/2016/6/60/T--ShanghaitechChina--chanqingzhuangzhizuizhong.png" style="width:72%"></center></div>
+  <div class="col-lg-6">
+<center><img src="https://static.igem.org/mediawiki/2016/0/06/T--ShanghaitechChina--Hydrogenase--chanqingzhuangzhixijie.png" style="width:72%"></center>
+</div>
+ <div class="col-lg-12">
+<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
-<center><img src="https://static.igem.org/mediawiki/2016/6/60/T--ShanghaitechChina--chanqingzhuangzhizuizhong.png" style="width:80%"></center>
+data and generate the curve of concentration variation within a period of time. <p></p>
-<p style="text-align:center"><b>Figure 1</b> Apparatus of the hydrogen production assay.</p>
+<p id="AResults" style="margin-bottom:80px"></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>
+<h3 >(3) Results</h3>
-<h3 id="AResults">Results</h3>
 <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>
@@ Line 46: / Line 78: @@
 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>
-As mentioned earlier, hydrogenase catalyzes the reversible oxidation of molecular hydrogen (H2). 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 7. 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.
+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>
-          <p style="text-align:center"><b>Figure 2</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>
           During the period under lighting, 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.<p></p>
 <h4><b>c) Hydrogen production with nano rods suspension replaced by nano rods bound to biofilm beads.</b></h4>
@@ Line 66: / Line 98: @@
           <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 (Figure4, 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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-<div id="p9" class="content">
+<p id="Potential use for wider application"></p>
+<div  class="content" >
+  <div class="row">
+    <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>
+<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 H_2 Production by Photocatalytic Decomposition of Water[J]. Imaging Science & Photochemistry, 2009, 27(3):225-232.</p>
+<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 H2, Production of a Whole-Cell Reaction by Recombinant Escherichia coli, Cells Expressing [FeFe]-Hydrogenase and Maturases Genes[J]. Angewandte Chemie, 2016</p>
+<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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