Difference between revisions of "Team:KUAS Korea/Experiments"

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                        <img src="https://static.igem.org/mediawiki/2016/d/d2/Korea_U_Seoul_figure1.jpeg">
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  <img src="https://static.igem.org/mediawiki/2017/a/a9/T--KUAS_Korea--HrtR.png">
 
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<h4>1.Preparing Agar degrading Enzymes</h4><br>
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<h4>1. Designing the Heme sensing device </h4><br>
  
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<p><font size=4>We used 3 enzymes to degrade agar in our device which are, Agarase, NABH (Neoagarobiose hydrolase), and AHGD (anhydrogalactose dehydrogenase).
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<p><font size=4>We have looked for the precedent researches on heme resistance of gut microbiome, we have decided to select the external detector system using HssS & HssR protein and the internal dectector utilizing HrtR protein. We designed a complete set comprised of promoter, sensing protein, and marker by inserting genetic marker instead of the efflux pump in the conventional mechanism.</font></p>
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All of the enzymes are displayed on the surface of <em>E.coli</em> BW25113 using <em>E.coli</em> surface display vector pATLIC.
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These enzymes were already cloned and evaluated in theses. So we did not have to evaluate them ourselves. Related theses are listed on the reference list.  
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One of the enzymes, NABH works as dimers, so it is not functional on the surface of <em>E.coli</em>. 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 <em>E.coli</em> culture, TEV will cut the TEV site and the displayed protein will be ‘secreted’.
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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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<h4>2. <em>Shewanella oneidensis</em> MR-1</h4><br>
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<h4>2. Constructing the Heme sensing device</h4><br>
  
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<p><font size=4><em>Shewanella oneidensis</em> 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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<p><font size=4>➀ In the case of HssS, HssR, we could find the matching sequence in Bacillus cereus ATCC 14579 by BLASTing the sequence that was mentioned in the former research. We received this bacteria from Korean Collection of Type Culture(KCTC), cultured them and collected a single colony.  We extracted the gDNA of B.cereus, and added igem prefix, suffix respectively to the front and end of these HssR, HssS sequences. We also conducted PCR of gDNA template using this primer we designed.</p><br>
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<p><font size=4>➁ We modified HrtR and its promoter by adding iGEM prefix at the front, and suffix at the end. Moreover, we ordered from the IDT as a gBlock. We inserted the arrived gBlock utilizing the 3A assemble and TA cloning. We also screened the bacteria with ones that do not show the expression of RFP. We then cleaved the cloning vector using restriction enzymes and processed gel electrophoresis to check if the part was correct inserted.</p>
  
 
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<h4>3. Preparing Diaphorase</h4><br>
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<h4>3. Transformation of the <em>Lactobacillus plantarum</em> L67</h4><br>
  
  
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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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<p>We received <em>L. plantarum</em> L67 from the lab of Professor Se Jong Oh, Chonnam University. Then, we cultured the bacteria on MRS media and collected single colony. Then we tested antibiotic resistance to prove that we cultured the appropriate bacteria. For the selection of transformation vector of <em>Lactobacillus</em>, we have chosen the gram-positive shuttle vector that our lab already owned. We made electron competent cell of <em>lactobacillus</em> and attempted transformation in various conditions with different DNA amount and voltage. However, the transformation was unfortunately not successful, and we concluded that the reason was the malfunction of replicon in the <em>Lactobacillus</em>. So we additionally ordered the Lactic Acid Bacteria (LAB) replicon for BBa_K1033206.</font> </p>
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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. </font> </p>
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<h4>4. Battery Device Validation</h4><br>
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<h4>4. Construction of the Screenable marker</h4><br>
  
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<p><h><strong>1) MFC</strong></h></p>
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<p>When defining the presence of occult blood in our feces, we thought it would be difficult for users to recognize if we used general GFP, due to the low fluorescence. Therefore, we planned to use chromoprotein rather than fluorescent proteins like GFP. We found out that 2013 Uppsala team previously used <em>Lactobacillus</em> and chromoprotein in their project. So we received the part this team submitted from the iGEM HQ and tested expression. </font>
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<p><font size=4>We measured the voltage that <em>Shewanella oneidensis</em> 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 <em>E. coli</em> 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. </font>
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<p><h><strong>2) EFC</strong></p></h>
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<p><font size=4>>We also validated our device as an EFC. For the validation, diaphorase expressed <em>E. coli</em> BL21(DE3) was lysed by sonication and was applied into the anode chamber of our battery device. Methylene blue was given as the mediator. Lysed BL21(DE3) with void vector was the control. NADH was added to both batteries. </font>
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<h4>5. EMFC Operation</h4><br>
 
  
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<p><font size=4>Our final goal was to prove that our device works as an EMFC.<em> Shewanella oneidensis</em> 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 without agar was set as the control. </font>
 
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Revision as of 08:22, 28 October 2017

Experiments

1. Designing the Heme sensing device


We have looked for the precedent researches on heme resistance of gut microbiome, we have decided to select the external detector system using HssS & HssR protein and the internal dectector utilizing HrtR protein. We designed a complete set comprised of promoter, sensing protein, and marker by inserting genetic marker instead of the efflux pump in the conventional mechanism.



2. Constructing the Heme sensing device


➀ In the case of HssS, HssR, we could find the matching sequence in Bacillus cereus ATCC 14579 by BLASTing the sequence that was mentioned in the former research. We received this bacteria from Korean Collection of Type Culture(KCTC), cultured them and collected a single colony. We extracted the gDNA of B.cereus, and added igem prefix, suffix respectively to the front and end of these HssR, HssS sequences. We also conducted PCR of gDNA template using this primer we designed.


➁ We modified HrtR and its promoter by adding iGEM prefix at the front, and suffix at the end. Moreover, we ordered from the IDT as a gBlock. We inserted the arrived gBlock utilizing the 3A assemble and TA cloning. We also screened the bacteria with ones that do not show the expression of RFP. We then cleaved the cloning vector using restriction enzymes and processed gel electrophoresis to check if the part was correct inserted.



3. Transformation of the Lactobacillus plantarum L67


We received L. plantarum L67 from the lab of Professor Se Jong Oh, Chonnam University. Then, we cultured the bacteria on MRS media and collected single colony. Then we tested antibiotic resistance to prove that we cultured the appropriate bacteria. For the selection of transformation vector of Lactobacillus, we have chosen the gram-positive shuttle vector that our lab already owned. We made electron competent cell of lactobacillus and attempted transformation in various conditions with different DNA amount and voltage. However, the transformation was unfortunately not successful, and we concluded that the reason was the malfunction of replicon in the Lactobacillus. So we additionally ordered the Lactic Acid Bacteria (LAB) replicon for BBa_K1033206.



4. Construction of the Screenable marker


When defining the presence of occult blood in our feces, we thought it would be difficult for users to recognize if we used general GFP, due to the low fluorescence. Therefore, we planned to use chromoprotein rather than fluorescent proteins like GFP. We found out that 2013 Uppsala team previously used Lactobacillus and chromoprotein in their project. So we received the part this team submitted from the iGEM HQ and tested expression.





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.