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Protein Trafficking
Responsible for:
Target ACE proteins into mitochondria
Parts submitted: BBa_K1119000, BBa_K1119001 & BBa_K1119009
Team:Hong Kong HKUST/modules
From 2013.igem.org
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| - | <br><br><br><div id="slide"><h3 class="title">Project Outline</h3><p id="isi">The design of our project is to build a constitutive and inducible glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate. First, the glyxylate shunt is introduced into mammalian cell by two bacterial native genes, aceA and aceB. However, unlike bacteria, citric acid cycle occurs in mitochondria for mammals. Thus, we translocated glyoxylate enzymes into mitochondria by fusing them with mitochondrial leader sequence. Lastly, for the glyoxylate enzymes to be expressed constitutively, we have fused them with mammalian constitutive promoters, namely CMV and EF-1alpha promoters. For inducible circuit, the glyoxylate enzymes are designed to be fused with fatty acid inducible promoters such as fatty acid binding protein promoter. <strong>Hover</strong> your mouse and <strong>click</strong> on the images below to learn more about each modules! | + | <br><br><br><div id="slide"><h3 class="title">Project Outline</h3><p id="isi">The design of our project is to build a constitutive and inducible glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate. First, the glyxylate shunt is introduced into mammalian cell by two bacterial native genes, aceA and aceB. However, unlike bacteria, citric acid cycle occurs in mitochondria for mammals. Thus, we translocated glyoxylate enzymes into mitochondria by fusing them with mitochondrial leader sequence. Lastly, for the glyoxylate enzymes to be expressed constitutively, we have fused them with mammalian constitutive promoters, namely CMV and EF-1alpha promoters. For inducible circuit, the glyoxylate enzymes are designed to be fused with fatty acid inducible promoters such as fatty acid binding protein promoter. <strong>Hover</strong> your mouse and <strong>click</strong> on the images below to learn more about each modules!</p></div> |
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Our ultimate goal is to build a ‘smart’ glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate when there is a surplus of energy. To achieve this, we would have to first build the shunt itself in mammalian cells, which consists of two bacterial enzymes, isocitrate lyase (AceA) and malate synthase (AceB). These two glyoxylate enzymes were tagged with mitochondrial leader sequence, so they could be directed to the compartment where the citric acid cycle could be effected on. The two enzymes were initially driven by constitutive promoters (CMV and EF-1alpha promoters), which put the shunt in a constantly “ON” state and burns calories regardless of the energy profile. To improve this, we are developing fatty acid responsive promoters, which, when used to regulate the glyoxylate enzymes, should dispense energy only when it is in excess.</p></div> | Our ultimate goal is to build a ‘smart’ glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate when there is a surplus of energy. To achieve this, we would have to first build the shunt itself in mammalian cells, which consists of two bacterial enzymes, isocitrate lyase (AceA) and malate synthase (AceB). These two glyoxylate enzymes were tagged with mitochondrial leader sequence, so they could be directed to the compartment where the citric acid cycle could be effected on. The two enzymes were initially driven by constitutive promoters (CMV and EF-1alpha promoters), which put the shunt in a constantly “ON” state and burns calories regardless of the energy profile. To improve this, we are developing fatty acid responsive promoters, which, when used to regulate the glyoxylate enzymes, should dispense energy only when it is in excess.</p></div> | ||
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Revision as of 16:07, 27 September 2013
Project Outline
The design of our project is to build a constitutive and inducible glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate. First, the glyxylate shunt is introduced into mammalian cell by two bacterial native genes, aceA and aceB. However, unlike bacteria, citric acid cycle occurs in mitochondria for mammals. Thus, we translocated glyoxylate enzymes into mitochondria by fusing them with mitochondrial leader sequence. Lastly, for the glyoxylate enzymes to be expressed constitutively, we have fused them with mammalian constitutive promoters, namely CMV and EF-1alpha promoters. For inducible circuit, the glyoxylate enzymes are designed to be fused with fatty acid inducible promoters such as fatty acid binding protein promoter. Hover your mouse and click on the images below to learn more about each modules!
Version 2: Our ultimate goal is to build a ‘smart’ glyoxylate shunt that increases energy metabolism by accelerating fatty acid uptake rate when there is a surplus of energy. To achieve this, we would have to first build the shunt itself in mammalian cells, which consists of two bacterial enzymes, isocitrate lyase (AceA) and malate synthase (AceB). These two glyoxylate enzymes were tagged with mitochondrial leader sequence, so they could be directed to the compartment where the citric acid cycle could be effected on. The two enzymes were initially driven by constitutive promoters (CMV and EF-1alpha promoters), which put the shunt in a constantly “ON” state and burns calories regardless of the energy profile. To improve this, we are developing fatty acid responsive promoters, which, when used to regulate the glyoxylate enzymes, should dispense energy only when it is in excess.
Cell Viability & Fatty Acid Quantification
Responsible for:
Measuring cell viability at different fatty acid concentration & measure fatty acid uptake rate
Parts submitted: -
Fatty Acid Sensing Mechanism
Responsible for:
Introduce inducible system that allows tunable fatty acid uptake by sensing fatty acid concentration
Parts submitted: -
Glyoxylate Shunt
Responsible for:
Introduce glyoxylate enzymes responsible for the shunt
Parts submitted:BBa_K1119002, BBa_K1119003, BBa_K1119004, BBa_K1119006 & BBa_K1119008
