LechenQian (Talk | contribs) |
|||
(4 intermediate revisions by 2 users not shown) | |||
Line 20: | Line 20: | ||
</li> | </li> | ||
</ul> | </ul> | ||
+ | </li> | ||
+ | <li> | ||
+ | <a href="#Potential use for wider application">Potential use for wider application</a> | ||
</li> | </li> | ||
<li> | <li> | ||
Line 75: | 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> | 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 (<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 | + | 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> | ||
Line 103: | Line 106: | ||
</div> | </div> | ||
+ | |||
+ | <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> | <p id="Reference"></p> | ||
<div class="content"> | <div class="content"> | ||
Line 112: | Line 148: | ||
<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>[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 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>[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> | ||
</div></div> | </div></div> |
Latest revision as of 02:10, 20 October 2016