Difference between revisions of "Team:UrbanTundra Edmonton/Proof"

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<h1>The Bio Reaction</h1>
 
<h1>The Bio Reaction</h1>
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<h2>Our first attempt at Oxygen generation</h2>
 
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<span style="font-style: italic">Experimental Design:</span>
 
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  To generate oxygen in our system, from plasmid-borne Clds in E. coli, first requires the addition of three components as discussed under Project Description. IPTG is required to induce Cld gene expression. Fe<sup>2+</sup> and levulinic acid are required as precursors for heme synthesis which functions as a catalytic co-factor in the Cld enzyme. Since heme is synthesized in the cytoplasm we expected to see O<sub>2</sub> produced in both Cld(SP+) and Cld(SP-) strains. We hoped that this experiment would shed light on relatively efficiency and capacity of O<sub>2</sub> in the periplasm Cld(SP+) relative to the cytosol. Following gene expression, chlorite would be added to a final concentration of 0.1M where we expected to see the generation of bubbles.
 
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<span style="font-style: italic">Method:</span>
 
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Six 5 mL LB overnight cultures were set up using single colonies that were picked from the first 3 of each of the Cld-containing candidates  previously described in Fig.8 [Cld (SP+) 1,2 and 3; Cld(SP-) 1,2 and 3]. To each of the six cultures was added IPTG (1.0mM), levulinic acid (2.0mM) FeSO<sub>4</sub> (2.0mM). These concentrations approximate the concentrations reported by Thorell et al. (2003). Cells were incubated overnight at 37<sup>O</sup> C in a shaking incubator. The next day, each 5mL culture was pelleted and resuspended in 0.4mL of 10mM Tris, 1mM EDTA pH 8.0. The resuspended pellets were then transferred to plastic cuvettes as shown below in Fig.12). Chlorite (0.1M) was then added for a final concentration of 0.5M. </p>
 
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<span style="font-style: italic">Results and Discussion:</span>
 
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  Cultures expressing Cld(SP-) were noticeably redish brown in colour whereas those derived from Cld(SP+) showed the same colour of  conventional E coli. This suggest that heme-containing Cld(SP-) is being produced whereas heme-containing Cld(SP+) is not being produced. When chlorite was added to the resuspended cell pellets, the CLD(SP-) samples began to bubble almost instantaneously. In contrast, no bubbling was observed in the Cld(SP+) samples. The failure of  Cld(SP+) to produce O2 is not clear. It might have resulted from the ala to ser mutation revealed from the gene sequence or alternatively the failure of the non-native MalE signal peptide
 
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  <img src="https://static.igem.org/mediawiki/2016/c/c0/T--UrbanTundra_Edmonton--8.png" alt="Before and after Chlorite" max width="100%">
 
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  <h5>Figure 12</h5>
 
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<h4>Scaling up Oxygen Generation</h4>
 
<h4>Scaling up Oxygen Generation</h4>
 
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   We repeated the above experiment at larger scale with the following modifications. 50 mL cultures were grown overnight in 200mL Erlenmeyer flasks using the same conditions described above. In place of Cld(SP+) we used a culture of E. coli that contained no plasmid as a negative control. The next day stir bars were added to each flask and the cultures were stirred at moderate speed. Solid chlorite was added to each flask to a final concentration of 0.5M. A condom was placed over each flask to collect the generated O<sub>2</sub> . Condoms were selected because of their thin, flexible nature that when filled with O<sub>2</sub>  would produce  an approximate cylinder whose volume could be calculated from it diameter and length. The results are presented in the video in this <a href="2016.igem.org/Team:UrbanTundra_Edmonton/Proof">here</a>. 
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   We repeated the above experiment at larger scale with the following modifications. 50 mL cultures were grown overnight in 200mL Erlenmeyer flasks using the same conditions described above. In place of Cld(SP+) we used a culture of E. coli that contained no plasmid as a negative control. The next day stir bars were added to each flask and the cultures were stirred at moderate speed. Solid chlorite was added to each flask to a final concentration of 0.5M. A condom was placed over each flask to collect the generated O<sub>2</sub> . Condoms were selected because of their thin, flexible nature that when filled with O<sub>2</sub>  would produce  an approximate cylinder whose volume could be calculated from it diameter and length. The results are presented in the video in the video below.
 
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Revision as of 21:33, 27 November 2016


Urban Tundra | Intelligent Innovation

The Bio Reaction

Scaling up Oxygen Generation

We repeated the above experiment at larger scale with the following modifications. 50 mL cultures were grown overnight in 200mL Erlenmeyer flasks using the same conditions described above. In place of Cld(SP+) we used a culture of E. coli that contained no plasmid as a negative control. The next day stir bars were added to each flask and the cultures were stirred at moderate speed. Solid chlorite was added to each flask to a final concentration of 0.5M. A condom was placed over each flask to collect the generated O2 . Condoms were selected because of their thin, flexible nature that when filled with O2 would produce an approximate cylinder whose volume could be calculated from it diameter and length. The results are presented in the video in the video below.

Concept Sketch
  1. Martian soil and water are mixed together to create a sludge in this first module.
  2. The Martian soil solution is filtered through to the next stage where the perchlorate ions are absorbed by rods filled with activated charcoal.
  3. The activated charcoal rods are shunted into a module with boiling water. Boiling the water releases the perchlorate ions creating a solution of perchlorate ions. The solution is also diluted if necessary at this stage if the perchlorate concentration is too high, to ensure the survival of the E. Coli in the next stage.
  4. The perchlorate solution is precipitated into a chamber layered with the genetically modified E. Coli where the perchlorate is converted into oxygen and chloride ions. Biomass from the mission is used to feed the E. Coli, and the waste can be reused as fertilizer for possible agriculture operations. The oxygen gas and chloride is separated by a membrane after exiting the chamber.

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