Difference between revisions of "Team:UC San Diego/Description"

 
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<h3>Uses of Luciferase</h3>
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<p>The Lux system’s bioluminescent reaction has been the subject of intense research. It is most often used as a reporter for gene expression and for tracking animal cells <i>in vivo</i>, and has more recently been utilized in synthetic biology for biosensor construction. As a result, it offers a powerful tool to researchers seeking to monitor a wide variety of biological and chemical processes.</p>
  
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<h3>About the Lux System</h3>
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<p>The luminescent output of the Lux system is jointly controlled by the activity of luciferase and a substrate-producing enzymatic complex. Firefly luminescence is the most well-known example of this type of reaction, but bioluminescent reactions also occur in beetles, marine bacteria, and other organisms. Between species, these reactions differ mechanistically and involve structurally distinct substrates and enzymes. We are studying the bacterial Lux system, in which an enzyme complex converts fatty acids into an aldehyde. Catalyzed by luciferase, these aldehydes then react with FMNH<sub>2</sub> and oxygen, emitting a photon and producing a fatty acid, FMN, and water. FMNH<sub>2</sub> is then regenerated by a flavin reductase, allowing for continuous light production with oxygen input.</p>
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<h3>Our Approach</h3>
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<p>Using existing approaches, mathematical models are limited in how they can predict and optimize metabolic pathways. We will be expressing several permutations of the bacterial Lux system to empirically determine its rate limiting steps by measuring the resulting light production. The identification of rate-limiting steps will then allow us to optimize the pathway. With these findings, an improved and experimentally validated mathematical model of the pathway will be constructed. By strategically altering biosynthetic gene expression, we will gain both a generalizable method to rapidly optimize metabolic pathways and a means to tailor the reporter/sensor to better suit our needs (i.e. make it brighter).</p>
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            <h2>Project Description</h2>
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<p>The lux system’s characteristic bioluminescent reaction has been the subject of intense research due to the potential of luciferase as a reporter. Compared to common fluorescent reporter systems, luciferase assays offer increased sensitivity and do not run the risk of inducing phototoxicity in the system being analyzed. As a result, they offer a powerful tool to researchers seeking to monitor a wide variety of biological processes. However, the environmental conditions, such as the amount of metabolites available, affects the bioluminescent reaction. Therefore, our objective is to modify the expression of the genes lux C, D, and E to determine the rate-limiting steps.</p>
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<p>We will develop and assemble three plasmids that expressed the aforementioned genes in a 1:1:2 ratio.. The plasmid vectors will be inserted into Saccharomyces cerevisiae, and the variation in light yield will be measured. To further our findings, we will create an experimentally validated mathematical model that provides an in depth in silico analysis. With these results, we can optimize the pathway by controlling the expression of certain enzymes.</p>
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Latest revision as of 20:54, 20 November 2015

post image

Uses of Luciferase

The Lux system’s bioluminescent reaction has been the subject of intense research. It is most often used as a reporter for gene expression and for tracking animal cells in vivo, and has more recently been utilized in synthetic biology for biosensor construction. As a result, it offers a powerful tool to researchers seeking to monitor a wide variety of biological and chemical processes.

About the Lux System

The luminescent output of the Lux system is jointly controlled by the activity of luciferase and a substrate-producing enzymatic complex. Firefly luminescence is the most well-known example of this type of reaction, but bioluminescent reactions also occur in beetles, marine bacteria, and other organisms. Between species, these reactions differ mechanistically and involve structurally distinct substrates and enzymes. We are studying the bacterial Lux system, in which an enzyme complex converts fatty acids into an aldehyde. Catalyzed by luciferase, these aldehydes then react with FMNH2 and oxygen, emitting a photon and producing a fatty acid, FMN, and water. FMNH2 is then regenerated by a flavin reductase, allowing for continuous light production with oxygen input.

Our Approach

Using existing approaches, mathematical models are limited in how they can predict and optimize metabolic pathways. We will be expressing several permutations of the bacterial Lux system to empirically determine its rate limiting steps by measuring the resulting light production. The identification of rate-limiting steps will then allow us to optimize the pathway. With these findings, an improved and experimentally validated mathematical model of the pathway will be constructed. By strategically altering biosynthetic gene expression, we will gain both a generalizable method to rapidly optimize metabolic pathways and a means to tailor the reporter/sensor to better suit our needs (i.e. make it brighter).