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Team:Wisconsin-Madison
From 2012.igem.org
Creating a tool to evaluate the translation of heterologous genes in Eschericia coli.
In synthetic biology, a powerful method for the production of novel metabolites is the expression of heterologous genes in Escherichia coli. 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 troubleshoot the expression of limonene synthase, an enzyme that catalyzes the production of limonene, a monoterpene with potential as a renewable jet fuel.
Why Produce Limonene?
The goal of this project is to engineer Escherichia coli to produce a compound called limonene. Limonene is a 10 carbon monoterpene and is found naturally in the oils of citrus fruits. It is used as a cleaning agent, solvent, food additive and is even finding a place in new medical applications. Limonene also possesses the chemical properties of an ideal biofuel, sparking interest specifically in its application as a jet fuel due to its low freezing point. Currently, we are limited to extracting limonene directly from citrus fruits which prevents its collection in quantities large enough to be useful as a biofuel. E coli. could be used to produce limonene more effectively and efficiently.
Why translational coupling?
In the beginning of the summer our team’s goal was to produce a compound called limonene. All of our strains failed to produce limonene, and we were unsure of the cause. One hypothesis was that there was a problem in the translation of limonene synthase. To test this hypothesis, we used a translational coupling cassette to determine if limonene synthase was even being translated, or if the problem was elsewhere in the cell.
