Difference between revisions of "Team:Korea U Seoul/Experiments"

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<div class="container-fluid page-heading" style="background-image: url(https://static.igem.org/mediawiki/2016/5/53/Group.jpg)">
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    <h3>Experiments</h3>
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            <div class="section" id="design">
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
 
  
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<h4>1.Preparing Agar degrading Enzymes</h4>
  
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<p>We used 3 enzymes to degrade agar in our device which are, Agarase, NABH (Neoagarobiose hydrolase), and AHGD (anhydrogalactose dehydrogenase).
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<h5>What should this page contain?</h5>
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All of the enzymes are displayed on the surface of E.coli BW25113 using E.coli surface display vector pATLIC.
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<ul>
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These enzymes were already cloned and evaluated in theses. So we did not have to evaluate them ourselves.
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<li> Protocols </li>
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One of the enzymes, NABH works as dimers, so it is not functional on the surface of E.coli. However, our surface display system can solve this problem by adding TEV protease. TEV protease site exist in our surface display vector pATLIC. If you add TEV  into the surface displayed E.coli culture, TEV will cut the TEV site and the displayed protein will be ‘secreted’.
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<li> Experiments </li>
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TEV was already cloned in pET vector in our lab, so we also did not have to clone it for ourselves. TEV was expressed in BL21(DE3).
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<li>Documentation of the development of your project </li>
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<h5>Inspiration</h5>
 
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
 
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
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<h4>2. Shewanella oneidensis MR-1</h4>
  
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<p>Shewanella oneidensis MR-1 is a bacteria that can reduce metal instead of oxygen, thus generating electricity in a battery device. We did not need any molecular work for this bacteria since it already has the function we need for our project. </p>
  
  
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<h4>3. Preparing Diaphorase</h4>
  
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<p>Diaphorase is a type of enzyme that can generate electricity using NAD(P)H as the source of electron.
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The gene of the diaphorase is from sus scrofa, which is pig. It has been codon optimized and synthesized. We then used it to clone it into an expression vector pB3, and it was expressed in BL21(DE3).
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It was then tested in diaphorase assay to check its activity. </p>
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<h4>4. Battery Device Validation</h4>
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<p>1) MFC</p>
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<p>We measured the voltage that Shewanella oneidensis MR-1 produce when formate is given since MR-1 uses formate to generate electricity. As controls, we measured the voltage of the battery with E. coli BW25113 in the anode chamber, and the battery without microbes. Methylene blue was given as the mediator. The purpose of this experiment is to make sure that our battery device works as an MFC.
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</p>
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<p>2) EFC</p>
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We also perform a validated our device as an EFC. For the test, diaphorase expressed E. coli BL21(DE3) was lysed by sonication and was applied into the anode chamber of our battery device. Methylene blue was given as the mediator. BL21(DE3) with void vector was the control.
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<h4>5. EMFC validation</h4>
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<p>Our final validation was to prove that our device can be used as an EMFC. Shewanella oneidensis MR-1, BW25113 with displayed agar degrading enzymes, cell lysate of diaphorase expressed BL21(DE3), and cell lysate of TEV expressed BL21(DE3) was put into the anode chamber. Agar was used as the substrate. The battery device with only MFC pathway (Agarase, NABH, MR-1, and TEV) and the battery device with only EFC pathway (Agarase, NABH, AHGD, Diaphorase, and TEV) were set as controls. </p>
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<h4>References</h4>
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<ol class="references">
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<li>Yun, Eun Ju, et al. "Production of 3, 6-anhydro-L-galactose from agarose by agarolytic enzymes of Saccharophagus degradans 2-40." Process biochemistry 46.1 (2011): 88-93.</li>
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<li>Yun, Eun Ju, et al. "The novel catabolic pathway of 3, 6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium." Environmental microbiology 17.5 (2015): 1677-1688.</li>
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<li>Ko, Hyeok-Jin, et al. "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL." Applied and environmental microbiology 78.9 (2012): 3051-3058.</li>
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<li>Wang, Victor Bochuan, et al. "Metabolite-enabled mutualistic interaction between Shewanella oneidensis and Escherichia coli in a co-culture using an electrode as electron acceptor." Scientific reports 5 (2015).</li>
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<li>Zhu, Zhiguang, et al. "A high-energy-density sugar biobattery based on a synthetic enzymatic pathway." Nature communications 5 (2014).</li>
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<li>Watson, Valerie J., and Bruce E. Logan. "Power production in MFCs inoculated with Shewanella oneidensis MR‐1 or mixed cultures." Biotechnology and bioengineering 105.3 (2010): 489-498.</li>
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Revision as of 01:43, 11 October 2016

Experiments

1.Preparing Agar degrading Enzymes

We used 3 enzymes to degrade agar in our device which are, Agarase, NABH (Neoagarobiose hydrolase), and AHGD (anhydrogalactose dehydrogenase). All of the enzymes are displayed on the surface of E.coli BW25113 using E.coli surface display vector pATLIC. These enzymes were already cloned and evaluated in theses. So we did not have to evaluate them ourselves. One of the enzymes, NABH works as dimers, so it is not functional on the surface of E.coli. However, our surface display system can solve this problem by adding TEV protease. TEV protease site exist in our surface display vector pATLIC. If you add TEV into the surface displayed E.coli culture, TEV will cut the TEV site and the displayed protein will be ‘secreted’. TEV was already cloned in pET vector in our lab, so we also did not have to clone it for ourselves. TEV was expressed in BL21(DE3).

2. Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a bacteria that can reduce metal instead of oxygen, thus generating electricity in a battery device. We did not need any molecular work for this bacteria since it already has the function we need for our project.

3. Preparing Diaphorase

Diaphorase is a type of enzyme that can generate electricity using NAD(P)H as the source of electron. The gene of the diaphorase is from sus scrofa, which is pig. It has been codon optimized and synthesized. We then used it to clone it into an expression vector pB3, and it was expressed in BL21(DE3). It was then tested in diaphorase assay to check its activity.

4. Battery Device Validation

1) MFC

We measured the voltage that Shewanella oneidensis MR-1 produce when formate is given since MR-1 uses formate to generate electricity. As controls, we measured the voltage of the battery with E. coli BW25113 in the anode chamber, and the battery without microbes. Methylene blue was given as the mediator. The purpose of this experiment is to make sure that our battery device works as an MFC.

2) EFC

We also perform a validated our device as an EFC. For the test, diaphorase expressed E. coli BL21(DE3) was lysed by sonication and was applied into the anode chamber of our battery device. Methylene blue was given as the mediator. BL21(DE3) with void vector was the control.

5. EMFC validation

Our final validation was to prove that our device can be used as an EMFC. Shewanella oneidensis MR-1, BW25113 with displayed agar degrading enzymes, cell lysate of diaphorase expressed BL21(DE3), and cell lysate of TEV expressed BL21(DE3) was put into the anode chamber. Agar was used as the substrate. The battery device with only MFC pathway (Agarase, NABH, MR-1, and TEV) and the battery device with only EFC pathway (Agarase, NABH, AHGD, Diaphorase, and TEV) were set as controls.

References

  1. Yun, Eun Ju, et al. "Production of 3, 6-anhydro-L-galactose from agarose by agarolytic enzymes of Saccharophagus degradans 2-40." Process biochemistry 46.1 (2011): 88-93.
  2. Yun, Eun Ju, et al. "The novel catabolic pathway of 3, 6‐anhydro‐L‐galactose, the main component of red macroalgae, in a marine bacterium." Environmental microbiology 17.5 (2015): 1677-1688.
  3. Ko, Hyeok-Jin, et al. "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia coli with a novel ligation-independent cloning vector based on the autotransporter YfaL." Applied and environmental microbiology 78.9 (2012): 3051-3058.
  4. Wang, Victor Bochuan, et al. "Metabolite-enabled mutualistic interaction between Shewanella oneidensis and Escherichia coli in a co-culture using an electrode as electron acceptor." Scientific reports 5 (2015).
  5. Zhu, Zhiguang, et al. "A high-energy-density sugar biobattery based on a synthetic enzymatic pathway." Nature communications 5 (2014).
  6. Watson, Valerie J., and Bruce E. Logan. "Power production in MFCs inoculated with Shewanella oneidensis MR‐1 or mixed cultures." Biotechnology and bioengineering 105.3 (2010): 489-498.