Team:Wisconsin-Madison

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       <p class = "classtheoverview"> Creating a tool to evaluate the translation of heterologous genes in Eschericia coli. </p>
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       <p class = "classtheoverview"> <strong> The Translational Coupling Cassette: a tool for evaluating the translation of heterologous proteins in <i>Escherichia coli</i>. </strong></p>
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      <p class = "classtheoverview"> Engineering the limonene production metabolic pathway: TCC. </p>
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       <p align="left" class = "classtheinlinecontent2"> A powerful method for the production of novel metabolites is the expression of heterologous enzymes in a bacterial host. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion-compatible system for testing the translation of a gene of interest. This system couples the translation of the target gene to a fluorescent reporter gene; fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to optimize the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.</p>
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      <p align="left" class="classtheinlinecontent"> <strong style="font-style:italic; color: rgb(183, 1, 1);">TCC</strong>
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       <p align="left" class="classtheinlinecontent">Limonene is a colorless, liquid hydrocarbon that smells like lemon. It is found in the oils of citrus fruits and is primarily used as a cleaning agent, solvent, and flavorful food additive. Limonene also possesses chemical properties that make it an ideal jet fuel due to it's low freezing point and high combustibility. E. coli already has part of the metabolic pathway required to produce limonene, however we will be inserting genes from S. cerevisiae to create the mevalonate pathway in the cell. We will also codon optimize the Limonene Synthase (LimS1) gene which is the final step in converting GPP to limonene. Gas chromatography/mass spectrometry will be used to determine how much limonene the E. coli is producing. </p>
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Latest revision as of 20:08, 26 October 2012


The Translational Coupling Cassette: a tool for evaluating the translation of heterologous proteins in Escherichia coli.

A powerful method for the production of novel metabolites is the expression of heterologous enzymes in a bacterial host. A common challenge when using non-native genes in metabolic engineering is determining if they are being properly expressed. To address this issue, we have constructed a BioFusion-compatible system for testing the translation of a gene of interest. This system couples the translation of the target gene to a fluorescent reporter gene; fluorescence will only be detected when the target gene is entirely translated. This construct enables synthetic biologists to quickly determine if a gene is being expressed without the need for costly antibodies or analytical instruments (e.g. mass spectrometry). Currently, we are utilizing this cassette to optimize the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.









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