Difference between revisions of "Team:ShanghaitechChina/Proof"

Revision as of 01:58, 19 October 2016

igem2016:ShanghaiTech

Abstract

Solar Hunter is an artificial hydrogen production system comprising biofilm-anchored semiconductor nanorods (NRs) which efficiently convert photons to electrons, and engineered strain expressing [FeFe] hydrogenase that can efficiently catalyze Hydrogen production upon receiving the electrons donated by NRs. The success of this integrative hydrogen-producing system relies on robust construction and functional characterization of each part separately. We have proved that we successfully constructed and characterized our components, as revealed below.

For the full demonstration of the system with all the components, please refer to Demonstration of our Work

Main points we achieved in our project

1. Successful synthesis and characterization of CdS Nanorods.

2. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA-Histag fused protein allowed firm binding of semiconductor nanomaterials. BioBrick BBa_K2132001

3. Successful integration of [FeFe]-hydrogenase gene clusters from Clostridium acetobutylicum into one single plasmid to allow reliable expression.

1. Successful hydrogen production with freely-flowing CdS Nanorods.

Detailed Proof

a. We, first, need to obtain a suitable nanomaterial, CdS, that absorbs solar energy and convert it into electrons. The detailed synthesis and characterization data is shown in our webpage Nanomaterials Session for further information. Some key data are reproduced below.

Synthesis and Characterization

Results

Synthesis and Characterization of CdS Nanorods

We synthesized CdS nanorods following a procedure adapted from a previously published protocol[1]. The synthesis procedure mainly contains two steps: synthesis of CdS seeds, followed by growth of CdS nanorods using CdS nanoparticles as nuclei.

Figure 3. Solutions of CdS nanoparticle seeds in TOP (left), CdS NRs in toluene (right)

Characterization of UV-Vis was performed to calculate the concentration of CdS seed in TOP solution and CdS nanorod in toluene solution. Also, PL spectrum of CdS NRs in toluene was collected to investigate the emission attribute of the nanorod. TEM image of the nanorods was acquired to study the shape and size distribution.

Figure 4. Result of CdS NRs ligand exchange experiments.

A ligand exchange experiment was performed and the result is shown in Figure 4

Figure 5. UV-Vis spectra of CdS seeds in TOP (A) and CdS nanorods in toluene (B);. Photoluminescence Spectrum of CdS nanorods in toluene (C).

The concentration of CdS seeds and CdS NR products were determined by using the UV-Vis spectrometer (Fig. 5 A, B). The peak shown in PL spectrum (Fig. 5 C) matches the absorption peak of the UV-Vis spectra, which thus proves the synthesis of CdS NRs.

Figure 6. TEM images of CdS NRs (A) and size distribution of CdS NRs (B). Note: 100 NRs in total were measured to determine the size distribution. The NRs thus measured have an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.

TEM confirms that synthesized products show nanorod feature, with an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.

b. To allow easy recycling of precious semiconductor nanomaterials, we utilized engineered biofilms to anchor nanomaterials via metal coordination chemistry. Please refer to Biofilm Session for details of the successful construction and characterization of engineered biofilms that allow firm binding of nanomaterials. Key data are reproduced below.

First, the simplest design, CsgA-HisTag.

3.Quantum dots fluorescence test: successful binding test of Histag with nanomaterials (CdSeS/CdSe/ZnS core/shell quantum dots)

After confirming that our parts success in biofilm expression, we are going to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles. We apply suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 1h incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and QDs are attached with biofilms, thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgA-Histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow. The picture was snapped by ChemiDoc MP,BioRad, false colored.

Fig 5. Fluorescence test of CsgA-His binding with nanomaterials

4. TEM: visualization of binding test

Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. The first image illustrates biofilm areas which are densely covered by QDs after induced for 72h and incubated, compared to the second image which is not incubated with nanoparticles CdSe. The third one is a negative control without inducer, bacteria scattered without forming biofilm

Fig 6.Representative TEM images of biotemplated CdS quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are densely attached to biofilm.

Finally, transmission electron microscopy(TEM) visualize the microscopic binding effect of CsgA-Histag fused biofilm with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. From the first picture, it shows biofilm areas are densely covered by CdS nanorods. As can be clearly seen from the second figure, with inducer, there is distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. Thus we ultimately confirm the viability of bio-abiotic hybrid system.

Fig 7.Representative TEM images of biotemplated CdS nanorods on CsgA-His.

Second, The complex design with extra function of binding SpyTag-linked enzymes in addition to its nanomaterial-binding through HisTag. This is realized with our Part BBa_K2132001 under the promoter of tetO.

2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials

Then comes to the next part: we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify is the fluorescence assay of binding with nanomaterials.

Fig 9. Congo Red Assay of His-CsgA-SpyCatcher

His-CsgA-SpyCatcher-Histag

After applying the same steps as introduced above, the bottom of left well show a large area of bright fluorescence, manifesting His-CsgA-SpyCatcher-Histag mutant secreted biofilms under the control of inducer and Histags on it is not blocked by SpyCatcher protein. What is more, it is firmly attached with inorganic materials (i.e.quantum dots) through ligand. From this assay, we assure that the SpyCatcher will not impose negative effect on the binding between nanomaterial and biofilm. The picture was snapped by ChemiDoc MP, BioRad, false colored.

Fig 10. Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.

3. TEM: visualization of binding test

TEM further characterize the biofilm expressed by strains secreted His-CsgA-SpyCatcher-Histag (HSCH). The distinct nanofiber network manifests the large biofilm expression.

Fig 12. aTc induced secretion of His-CsgA-SpyCatcher-Histag visualized by TEM. Without the presence of inducer, there’s no nanofiber formation around scattered bacteria.

CsgA-His can interface with different inorganic materials since they form the coordinate bonds with the same ligand, Co-NTA, on nanomaterials. Here we use to AuNPs in place of quantum dots and nanomaterials to characterize the validity of Histags on CsgA fused amyloid protein and meanwhile prove the versatility of our biofilm-based platform. As the figures shown, we confirm the feasibility of our newly constructed biobricks to template inorganic material and thus form bio-abiotic hybrid system.

Fig 13. After aTc induced, biofilm secreted by His-CsgA-SpyCatcher-Histag organizes AuNP around the cells. In contrast with the one without inducer, where nothing was on the smooth outermembrane of bacteria.

c. Finally, high-activity hydrogenase is necessary for our system. To achieve efficient enzymatic activities, we codon-optimized and constructed the whole hydrogenase gene clusters (from Clostridium Acetobutylicum) by leveraging the multi-expression Acembl System. Please refer to Hydrogenase Session for more details.

Results

a) Hydrogen production system with free-flowing CdS nanorod.

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 (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.

Figure 2 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.

@@ Line 41: / Line 41: @@
-<div id="Abstract" class="content">
+<div id="Solar Hunter in a Nutshell" class="content">
    <div class="row">
      <div class="col-lg-12">
    <h1 align="center">Abstract</h1>
-<h2>Solar Hunter is an artificial hydrogen production system comprising biofilm-anchored semiconductor nanorods (NRs) which efficiently convert photons to electrons, and engineered strain expressing [FeFe] hydrogenase that can efficiently catalyze Hydrogen production upon receiving the electrons donated by NRs.   The success of this integrative hydrogen-producing system relies on robust construction and functional characterization of each part separately.   We have proved that we successfully constructed and characterized our parts, as revealed below.  </h2>
+<h2>Solar Hunter is an artificial hydrogen production system comprising biofilm-anchored semiconductor nanorods (NRs) which efficiently convert photons to electrons, and engineered strain expressing [FeFe] hydrogenase that can efficiently catalyze Hydrogen production upon receiving the electrons donated by NRs.   The success of this integrative hydrogen-producing system relies on robust construction and functional characterization of each part separately.   We have proved that we successfully constructed and characterized our components, as revealed below.  </h2> For the full demonstration of the system with all the components, please refer to <b><a href="https://2016.igem.org/wiki/index.php?title=Team:ShanghaitechChina/Demonstration">Demonstration of our Work</a></b>
 </div>
+<div id="Main points we achieved in our project" class="content">
+  <div class="row">
+    <div class="col-lg-12">
+  <h1 align="center">Main points we achieved in our project</h1>
+<h2>1. Successful synthesis and characterization of CdS Nanorods.</h2><p></p>
+<h2>2. Successful production and characterization of engineered Biofilms, demonstrating that engineered biofilms composed of CsgA-Histag fused protein allowed firm binding of semiconductor nanomaterials.  <b><a href="http://parts.igem.org/Part:BBa_K2132001">BioBrick BBa_K2132001</a></b>
+<h2>3. Successful integration of [FeFe]-hydrogenase gene clusters from Clostridium acetobutylicum into one single plasmid to allow reliable expression.</h2><p></p>
+<h2>1. Successful hydrogen production with freely-flowing CdS Nanorods.</h2><p></p>
 </div>
 </div>
@@ Line 176: / Line 186: @@
 <h2>c. Finally, high-activity hydrogenase is necessary for our system. To achieve efficient enzymatic activities, we codon-optimized and constructed the whole hydrogenase gene clusters (from Clostridium Acetobutylicum) by leveraging the multi-expression Acembl System.  Please refer to <b><a href="https://2016.igem.org/Team:ShanghaitechChina/Hydrogen">Hydrogenase Session</b></a> for more details. </h2><p></p>
+<h3 id="AResults">Results</h3>
+<h4><b>a) Hydrogen production system with free-flowing CdS nanorod.</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>
+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.<p></p>
+<center><img class="pic3x full" src="https://static.igem.org/mediawiki/2016/b/b1/T--ShanghaitechChina--asasy-conditon--success.png"></center>
+<p style="text-align:center"><b>Figure 2</b></p>
+<center><h3>click to enlarge the figure</h3></center>
+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 <em>E. coli</em> 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.<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>
+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.
+<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>
+         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>
+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.<p></p>
+<center><img src="https://static.igem.org/mediawiki/2016/4/4f/T--ShanghaitechChina--asasy-withfinalplan-bidirectlycat.png"></center>
 </div>
@@ Line 183: / Line 211: @@
-<div id="Main points we achieved in our project" class="content">
-  <div class="row">
-    <div class="col-lg-12">
-  <h1 align="center">Main points we achieved in our project</h1>
-<h2>1. Successful synthesis and characterization of CdS NRs and CdSe QDs with desired features.</h2><p></p>
-<h2>2. Successful production and characterization of Biofilms, and proved demonstration that engineered biofilms composed of CsgA-Histag fused protein allowed firm binding of semiconductor nanomaterials.  <b><a href="http://parts.igem.org/Part:BBa_K2132001">BioBrick BBa_K2132001</a></b>
-<h2>3. Successful construction and sequence of [FeFe]-hydrogenase gene clusters from Clostridium acetobutylicum and integrate all genes into one single plasmid to allow reliable expression.</h2><p></p>