Team:NTNU Trondheim/Project

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<p>CO<sub>2</sub> emissions have recieved a lot of attention in modern times, due to concerns that high emission levels are facilitating global warming. Consequently, a lot of research is focused on ways of reducing CO<sub>2</sub> emissions from industry, and ways of fixating atmospheric CO<sub>2</sub> at a greater than normal rate.</p>
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<p>Our project is attempting to produce a plasmid, which when placed inside photosynthetic bacteria, increases their rate of CO<sub>2</sub> fixation. In order to achieve this, we first need to construct BioBricks that allow inducible expression of non native genes in our chassis; <i>Synechocystis</i> sp. PCC 6803, when assembled.</p>
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The first thing we needed to do was to isolate the flanking sequences from <i>Synechocystis</i>. This was accomplished by use of 'colony PCR', where the genome of <i>Synechocystis</i> was used as a template. We designed primers so that we would only amplify our desired flanking sequences.
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After verifying that our PCR amplified flanking sequences had the right sequence, we wanted to test them. The flanking sequences were tested by ligating them to a vector backbone, with a Kanamycin resistance insert between the two flanking sequences. The resulting plasmid had the following parts, in order:
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This plasmid was transformed into Synechocystis, and the transformed cells were cultured in BG11 growth medium containing kanamycin. Growth of Synechocystis cells in this medium, along with band shift colony PCR (See figure below), confirmed to us that the plasmid had been successfully taken up and integrated into the host genome through homologous recombination.
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We wanted to see if transforming the gene <i>glucose oxidase</i> into <i>Synechocystis</i> would lead to an increased rate of carbon fixation pr. growth rate.
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To achieve this we constructed a plasmid with eight parts:
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<li>"Right flank" sequence</li>
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As growth in <i>Synechocystis</i> is very slow, we did not have time to test this plasmid, and so it will <i>not</i> be submitted as a composite BioBrick.
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Instead, the plasmid was split up into several BioBricks, so that each BioBrick contains one part of the composite plasmid. The BioBricks are as follows:
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<li><a href="http://parts.igem.org/Part:BBa_K1424001">Right flank</a>  (Cloned from <i>Synechocystis</i>)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1424000">Left flank</a> (Cloned from <i>Synechocystis</i>)</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1424003">Kanamycin resistance gene</a> (Cloned from <i>Synechocystis</i> )</li>
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<li><a href="http://parts.igem.org/Part:BBa_K1424004">Glucose Oxidase gene</a> (Synthesized by GenScript, codon optimized for <i>Synechocystis</i>.)</li>
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These BioBricks should provide teams with the means to use <i>Synechocystis</i> as a chassis in future iGEM competitions.
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<h3>Future efforts</h3>
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<p>Our candiate gene for increasing CO<sub>2</sub> the fixation rate is <i>glucose oxidase</i>, which is not originally present in <i>Synechocystis</i>. This gene encodes the enzyme Glucose Oxidase, which essentially reduces the oxygen concentration inside the cell. RuBisCO, the CO<sub>2</sub> fixating enzyme in photosynthetic organisms, has a high affinity for binding O<sub>2</sub>, which can interfere with CO<sub>2</sub> binding. Reducing O<sub>2</sub> concentrations could therefore lead to an increased rate of CO<sub>2</sub> fixation in <i>Synechocystis</i>.</p>
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<p>Future efforts should focus on transforming the composite plasmid containing all 6 BioBricks into Synechocystis, and testing the carbon fixation rate of the resulting transformants. Such testing requires specialized equipment that is able to measure minute differences in the partial pressure of CO<sub>2<sub>.</p>  
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project" style="text-decoration:none;color:#1C140D">PROJECT</a> </td>
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<tr><td  bgColor="#FEE5AD"></td> <td colspan="3" width="975px" bgColor="#FEE5AD" align="center"> <h3>Team Example's Project name! </h3></td> <td  bgColor="#FEE5AD"></td> </tr>
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<h3>Project Introduction</h3>
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<p>CO<sub>2</sub> emissions have recieved a lot of attention in modern times, due to concerns that high emission levels are facilitating global warming. Consequently, a lot of research is focused on ways of reducing CO<sub>2</sub> emissions from industry, and ways of fixating atmospheric CO<sub>2</sub> at a greater than normal rate.</p>
+
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<p>Our project is attempting to produce a BioBrick, which when placed inside photosynthetic bacteria, increases their rate of CO<sub>2</sub> fixation. In order to achieve this, we first need to construct a BioBrick that allows inducible expression of non native genes in our chassis; <a href="http://www.genome.jp/kegg-bin/show_organism?org=syn"><i>Synechocystis</i> sp. PCC 6803</a></p>
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<h4>Results</h4>
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<li>Result 1 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.</li>
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<li>Result 2 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.</li>  
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<li>Result 3 - Lorem ipsum ad his scripta blandit partiendo, eum fastidii accumsan euripidis in, eum liber hendrerit an.</li>
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<a href="#co2" style="text-decoration:none;color:#000000">CO<sub>2</sub> fixation BioBrick </a> </td>
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<a href="#testing" style="text-decoration:none;color:#000000">Testing CO<sub>2</sub> fixation rate </a> </td>
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<h4 id="reporter"> Reporter BioBrick</h4>
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<p>The first stage of this project consists of creating a reporter BioBrick in order to confirm that we have are able to induce the expression of foreign genes in <a href="http://www.genome.jp/kegg-bin/show_organism?org=syn"><i>Synechocystis</i> sp. PCC 6803</a>. This reporter BioBrick makes use of several other BioBricks, such as <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034</a> and <a href="http://parts.igem.org/Part:BBa_C0012">BBa_C0012</a>.</p>
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<p>Once the reporter plasmid has been successfully constructed, we will verify that it is working as intended by inducing the expression of a fluorescent protein contained on the plasmid. Once induced, the colonies of cells transformed with this plasmid should exhibit a color that is determined by the fluorescent protein. </p>
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<p>The plasmid is transformed into <i>Synechocystis</i> sp. PCC 6803 by homologous recombination. Contained on the plasmid are left- and right flanking sequences obtained from the <i>Synechocystis</i> genome. These flanking sequences undergo homologous with their sister sequences in the genome of <i>Synechocystis</i>, integrating the plasmid sequence into the genome. </p>
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<p> The reporter plasmid will use a fluorescent protein placed under the control of an indicuble promoter. This will allow us to easily verify that the inducible expression system works as intended inside <i>Synechocystis</i> sp. PCC 6803.</p>
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<h4 id="fixating"> CO<sub>2</sub> fixating BioBrick</h4>
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<p>After verifying that the expression system is working as intended in <i>Synechocystis</i>, we will replace the fluorescent protein gene with one that is predicted to increase the rate of carbon fixation in the organism. The way we will idenfity this gene is by use of metabolic modelling</p>
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<p>We had plans of collaborating with <a href="http://www.sintef.no/home/Environment/CO2-fangst-og-handtering/"> SINTEF CO<sub>2</sub>-capture and storage</a>, which were kind enough to offer us the use of their facilities and equipment. Doing our planned experiments in their facilities would have provided us with evidence for the effect of <i>glucose oxidase</i> on <i>Synechocystis'</i> carbon fixation rate.</p>
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Latest revision as of 22:08, 17 October 2014

Team:NTNU_Trondheim/notebook - 2014.igem.org

 

Team:NTNU_Trondheim/Home

From 2014.igem.org

NTNU Genetically Engineered Machines

SynECO2

Introduction

CO2 emissions have recieved a lot of attention in modern times, due to concerns that high emission levels are facilitating global warming. Consequently, a lot of research is focused on ways of reducing CO2 emissions from industry, and ways of fixating atmospheric CO2 at a greater than normal rate.

Our project is attempting to produce a plasmid, which when placed inside photosynthetic bacteria, increases their rate of CO2 fixation. In order to achieve this, we first need to construct BioBricks that allow inducible expression of non native genes in our chassis; Synechocystis sp. PCC 6803, when assembled.

Making a Synechocystis compatible vector

In order to start working with Synechocystis sp. PCC 6803, we have to construct a plasmid that will allow insertion of foreign genes into the Synechocystis genome. As Synechocystis does not retain inserted plasmids, transformations must make use of conjugation in order to make the insert part its genome. The flank sequences have been chosen such that they are homologous to a pair of sequences in a neutral site of the Synechocystis genome. When this plasmid is transformed into Synechocystis, the DNA between the two flank sequences is conjugated into its genome.

The first thing we needed to do was to isolate the flanking sequences from Synechocystis. This was accomplished by use of 'colony PCR', where the genome of Synechocystis was used as a template. We designed primers so that we would only amplify our desired flanking sequences.

After verifying that our PCR amplified flanking sequences had the right sequence, we wanted to test them. The flanking sequences were tested by ligating them to a vector backbone, with a Kanamycin resistance insert between the two flanking sequences. The resulting plasmid had the following parts, in order:

  • Left flank
  • Kanamycin resistance
  • Right flank
  • Backbone

This plasmid was transformed into Synechocystis, and the transformed cells were cultured in BG11 growth medium containing kanamycin. Growth of Synechocystis cells in this medium, along with band shift colony PCR (See figure below), confirmed to us that the plasmid had been successfully taken up and integrated into the host genome through homologous recombination.

We wanted to see if transforming the gene glucose oxidase into Synechocystis would lead to an increased rate of carbon fixation pr. growth rate.

To achieve this we constructed a plasmid with eight parts:

  • "Right flank" sequence
  • Constitutionally active promoter + RBS
  • LacI repressor gene
  • Kanamycin resistance
  • LacI inducible promoter + RBS
  • Glucose Oxidase
  • "Left flank" sequence
  • Plasmid backbone

As growth in Synechocystis is very slow, we did not have time to test this plasmid, and so it will not be submitted as a composite BioBrick.

Instead, the plasmid was split up into several BioBricks, so that each BioBrick contains one part of the composite plasmid. The BioBricks are as follows:


These BioBricks should provide teams with the means to use Synechocystis as a chassis in future iGEM competitions.

Future efforts

Our candiate gene for increasing CO2 the fixation rate is glucose oxidase, which is not originally present in Synechocystis. This gene encodes the enzyme Glucose Oxidase, which essentially reduces the oxygen concentration inside the cell. RuBisCO, the CO2 fixating enzyme in photosynthetic organisms, has a high affinity for binding O2, which can interfere with CO2 binding. Reducing O2 concentrations could therefore lead to an increased rate of CO2 fixation in Synechocystis.

Future efforts should focus on transforming the composite plasmid containing all 6 BioBricks into Synechocystis, and testing the carbon fixation rate of the resulting transformants. Such testing requires specialized equipment that is able to measure minute differences in the partial pressure of CO2.

We had plans of collaborating with SINTEF CO2-capture and storage, which were kind enough to offer us the use of their facilities and equipment. Doing our planned experiments in their facilities would have provided us with evidence for the effect of glucose oxidase on Synechocystis' carbon fixation rate.