Team:Lethbridge/Project/Compartamentalization
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=<font color="white">Lumazine Synthase Microcompartment for Compartmentalization= | =<font color="white">Lumazine Synthase Microcompartment for Compartmentalization= | ||
- | Our project this year is to use existing biological pathways and mechanisms for the cleaning contaminated water such as the tailings ponds. We are using the protein <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color=" | + | Our project this year is to use existing biological pathways and mechanisms for the cleaning contaminated water such as the tailings ponds. We are using the protein <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Catechol_Degradation"><font color="#00DC00"> catechol-2,3-dioxygenase</font></a></html> as the hub that other chemicals that are harmful to the environment breaks down into. With a working system of breaking down these harmful chemicals into biologically useful molecules there needs to be a way to apply the pathway for the cleaning of the contaminated tailings ponds. To apply the system to the tailings we can do so by isolating the pathway (enzymes) from the cell and applying it like a dry powder or by applying the bacteria directly to the tailings ponds water. |
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- | The compartment we are using is made up from a single protein (lumazine synthase or LS) that forms an icosahedron by assembling 60, 120 or 180 of the monomers and is found in <i>Aquifex aeolicus</i> (Seebeck <i>et al.</i>, 2006). This protein has been characterized and shown that it forms this structure with a cavity that is able to encapsulate other molecules. In the previous characterization it was shown that by selectively mutating five of the interior amino acids of the compartment to glutamate and by attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (<html><a href="https://2009.igem.org/Team:Lethbridge/Modeling" target="new"><font color=" | + | The compartment we are using is made up from a single protein (lumazine synthase or LS) that forms an icosahedron by assembling 60, 120 or 180 of the monomers and is found in <i>Aquifex aeolicus</i> (Seebeck <i>et al.</i>, 2006). This protein has been characterized and shown that it forms this structure with a cavity that is able to encapsulate other molecules. In the previous characterization it was shown that by selectively mutating five of the interior amino acids of the compartment to glutamate and by attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (<html><a href="https://2009.igem.org/Team:Lethbridge/Modeling" target="new"><font color="#00DC00">Lethbridge 2009 Modeling</font></a></html>) (Seebeck <i>et al.</i>, 2006). We will be using these features to selectively target our catechol-2,3-dioxygenase into the compartment. |
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- | To characterize the means of targeting the tagged protein we will be using another expression construct as shown in Figure 1 that contains the IPTG inducible LS that also has two fluorescent proteins – cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) – that are controlled by an arabinose induced inverter. This would allow us to selectively express the LS by adding IPTG and repressing the fluorescent protein expression by adding arabinose. We have chosen to work with CFP and YFP due their ability to undergo fluorescence resonance energy transfer (FRET) that will allow us to observe their colocalization within the LS microcompartment (for a general overview of FRET visit <html><a href="http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer" target="new"><font color=" | + | To characterize the means of targeting the tagged protein we will be using another expression construct as shown in Figure 1 that contains the IPTG inducible LS that also has two fluorescent proteins – cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) – that are controlled by an arabinose induced inverter. This would allow us to selectively express the LS by adding IPTG and repressing the fluorescent protein expression by adding arabinose. We have chosen to work with CFP and YFP due their ability to undergo fluorescence resonance energy transfer (FRET) that will allow us to observe their colocalization within the LS microcompartment (for a general overview of FRET visit <html><a href="http://en.wikipedia.org/wiki/Förster_resonance_energy_transfer" target="new"><font color="#00DC00">Wikipedia </font></a></html>or <html><a href="https://2009.igem.org/Team:Lethbridge/Project#The_Experiments" target="new"><font color="#00DC00">Lethbridge 2009 FRET</font></a></html>). By observing FRET within the microcompartment it will demonstrate our ability to selectively localize multiple proteins within it. |
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