Team:Wisconsin-Madison

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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"> <strong>Expression, Purification, and Quality Control of Gibson Enzymes </strong></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 = "classtheinlinecontent2"> Gibson assembly is a DNA cloning technique that was pioneered in 2009 by Daniel Gibson (Gibson, D.G. et al. Nat. Methods 343-345 (2009)). This technique utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. Gibson assembly is significantly simpler to perform than prior conventional methods, as it is carried out in a single step and affords greater flexibility than methods relying on the use of restriction enzymes. Because of its simplicity and versatility, it has quickly become a common technique in molecular biology across the world. However, the financial cost of the necessary enzymes can be quite expensive, potentially limiting the access of smaller research labs and primarily undergraduate institutions to this groundbreaking technique.</p>
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This summer we have gene synthesized, expressed, purified, and assayed the three enzymes involved in Gibson assembly. We have carefully documented our protocol for purifying these enzymes and tested their function relative to commercial versions of the enzymes. Lastly, we have combined our purifed enzymes in appropriate ratios to perform Gibson assembly. We are in the process of carefully vetting the efficacy of our “in-house” Gibson mastermix. </p>
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{|align="justify"
 
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|You can write a background of your team here.  Give us a background of your team, the members, etc.  Or tell us more about something of your choosing.
 
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|[[Image:Wisconsin-Madison_logo.png|200px|right|frame]]
 
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''Tell us more about your project.  Give us background.  Use this as the abstract of your project.  Be descriptive but concise (1-2 paragraphs)''
 
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|align="center"|[[Team:Wisconsin-Madison | Team Wisconsin-Madison]]
 
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!align="center"|[[Team:Wisconsin-Madison|Home]]
 
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!align="center"|[[Team:Wisconsin-Madison/Modeling|Modeling]]
 
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!align="center"|[[Team:Wisconsin-Madison/Safety|Safety]]
 
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!align="center"|[[Team:Wisconsin-Madison/Attributions|Attributions]]
 
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Latest revision as of 18:53, 9 August 2013


Expression, Purification, and Quality Control of Gibson Enzymes

Gibson assembly is a DNA cloning technique that was pioneered in 2009 by Daniel Gibson (Gibson, D.G. et al. Nat. Methods 343-345 (2009)). This technique utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. Gibson assembly is significantly simpler to perform than prior conventional methods, as it is carried out in a single step and affords greater flexibility than methods relying on the use of restriction enzymes. Because of its simplicity and versatility, it has quickly become a common technique in molecular biology across the world. However, the financial cost of the necessary enzymes can be quite expensive, potentially limiting the access of smaller research labs and primarily undergraduate institutions to this groundbreaking technique.

This summer we have gene synthesized, expressed, purified, and assayed the three enzymes involved in Gibson assembly. We have carefully documented our protocol for purifying these enzymes and tested their function relative to commercial versions of the enzymes. Lastly, we have combined our purifed enzymes in appropriate ratios to perform Gibson assembly. We are in the process of carefully vetting the efficacy of our “in-house” Gibson mastermix.


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