Team:NTNU Trondheim/Project

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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project">Background</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Project/Modelling">Modelling</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Notebook">Notebook</a>
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Notebook">Notebook</a>
<a href="https://2014.igem.org/Team:NTNU_Trondheim/Notebook" class="flyout-toggle"><span> </span></a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Protocols">Protocols</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Acknowledgements/Sponsors">Acknowledgements</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Notebook">Overview</a>
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<a href="https://2014.igem.org/Team:NTNU_Trondheim/Acknowledgements/Sponsors">Sponsors</a>
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<h6>Project description</h6>
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<h6>SynECO<sub>2</sub></h6>
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<h2 class="centered">Project Description</h2>
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<h2 class="centered">SynECO<sub>2</sub></h2>
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<h3>Making a Synechocystis compatible vector</h3>
<h3>Making a Synechocystis compatible vector</h3>
<p>
<p>
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In order to start working with <i>Synechocystis</i> sp. PCC 6803, we have to construct a plasmid that will allow insertion of foreign genes into the <i>Synechocystis</i> genome. As <i>Synechocystis</i> does not retain inserted plasmids, transformations must make use of conjugation in order to make the insert part its genome.
+
In order to start working with <i>Synechocystis</i> sp. PCC 6803, we have to construct a plasmid that will allow insertion of foreign genes into the <i>Synechocystis</i> genome. As <i>Synechocystis</i> 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 <i>Synechocystis</i> genome. When this plasmid is transformed into <i>Synechocystis</i>, the DNA between the two flank sequences is conjugated into its genome.
</p>
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<p>
<p>
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To achieve this we have constructed a plasmid with eight parts:
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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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</p>
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<p>
 +
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:
 +
<ul>
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<li>Left flank</li>
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<li>Kanamycin resistance</li>
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<li>Right flank</li>
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<li>Backbone</li>
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</ul><br>
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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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<img src="https://static.igem.org/mediawiki/2014/c/c0/NTNU_homologous_confirmation.png">
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</p>
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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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<p>
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To achieve this we constructed a plasmid with eight parts:
<ul>
<ul>
<li>"Right flank" sequence</li>
<li>"Right flank" sequence</li>
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<li>Kanamycin resistance</li>
<li>Kanamycin resistance</li>
<li>LacI inducible promoter + RBS</li>
<li>LacI inducible promoter + RBS</li>
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<li>Red fluorescent protein</li>
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<li>Glucose Oxidase</li>
<li>"Left flank" sequence</li>
<li>"Left flank" sequence</li>
<li>Plasmid backbone</li>
<li>Plasmid backbone</li>
</ul><br>
</ul><br>
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The flank sequences have been chosen such that they are homologous to a pair of sequences in a neutral site of the <i>Synechocystis</i> genome. When this plasmid is transformed into <i>Synechocystis</i>, the DNA between the two flank sequences is conjugated into its genome. By growing cultures in a selection medium containing Kanamycin, the insert is kept inside the genome, allowing us to express foreign genes inside the organism.
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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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Since we are submitting to the iGEM registry, the plasmid will be split up into several BioBricks which each contain one of the parts of the complete plasmid. The BioBricks are as follows:
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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:
<ul>
<ul>
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<li>Right flank (Cloned from <i>Synechocystis</i> )</li>
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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>Left flank (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>LacI inducible promoter + RBS (Codon optimized for <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 allow teams the means to use <i>Synechocystis</i> as a chassis in future iGEM competitions.
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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>Increasing the CO<sub>2</sub> fixation rate</h3>
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<h3>Future efforts</h3>
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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. Once a candidate gene has been detected, it will be cloned and transformed into <i>Synechocystis</i>.</p>
+
<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>One candiate gene 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>
+
<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>
 +
 
 +
<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.