Team:Cornell/Project

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~~==''' Overall project '''==~~

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Our project goal design is a essentially two parts. First, we would like to develop a genetic switch sensitive to specific wavelengths of visible light and use this gene expression system to be able to lyse bacterial cells solely with light. The genetic light sensor is based on Chris Voigt’s “Multichromatic Control of Gene Expression in Escherichia coli,” which uses visible green light at 532 nm to induce a specific gene expression. The system is composed of a light activated surface protein which autophosphorylates an intermediate chromophore, and a reporter protein which binds to a specific promoter and is activated by the chromophore. The genes to be expressed downstream of the promoter are a genetic lysis cassette developed by Prof. Young at Texas A&M which was derived from the lambda phage lysis genome. This lysis system is very useful because the incubation period after gene expression is on the order of 50 minutes, and the actual lysis occurs within a matter of one minute thereafter. Our hope is to have lysis within a known timeframe and is specific to the green light.

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The second part of the project is an interesting application for the light lysis system. We plan on developing an enzyme-studded microfluidic channel in which the enzymes for a known biosynthetic pathway are bound to the surface and organized within the channel in a linear, chronological order. This would allow for a solution saturated with the initial substrates of the pathway to be modified in order as they flow down the channel. This process would reduce unwanted side reactions, and greatly reduce the purification costs associated with producing biomolecules. The enzymes for the pathway would be slightly modified using an Avi-Tag so that they may be biotinylated by E. coli. These enzymes would be expressed in liquid bacterial cultures for each enzyme that also have been transformed with the light lysis plasmid. The cultures would then be exposed to the green light, and after the proper induction time, the culture would be pumped through the channel coated with streptavidin. The use of microfluidics is necessary to direct the flow of lysate to only the region designated for each enzyme. The benefit of using the light sensor system is the ability flow the cell culture through the channel as it lyses which minimizes the possible time for the enzymes to degrade. The simplicity of the lysis process allows for large bacterial cultures to be lysed with ease, allowing for the scalability, and automation of the construction process.

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-==''' Overall project '''==
-Our project goal design is a essentially two parts. First, we would like to develop a genetic switch sensitive to specific wavelengths of visible light and use this gene expression system to be able to lyse bacterial cells solely with light. The genetic light sensor is based on Chris Voigt’s “Multichromatic Control of Gene Expression in Escherichia coli,” which uses visible green light at 532 nm to induce a specific gene expression. The system is composed of a light activated surface protein which autophosphorylates an intermediate chromophore, and a reporter protein which binds to a specific promoter and is activated by the chromophore. The genes to be expressed downstream of the promoter are a genetic lysis cassette developed by Prof. Young at Texas A&M which was derived from the lambda phage lysis genome.  This lysis system is very useful because the incubation period after gene expression is on the order of 50 minutes, and the actual lysis occurs within a matter of one minute thereafter. Our hope is to have lysis within a known timeframe and is specific to the green light.
-The second part of the project is an interesting application for the light lysis system. We plan on developing an enzyme-studded microfluidic channel in which the enzymes for a known biosynthetic pathway are bound to the surface and organized within the channel in a linear, chronological order. This would allow for a solution saturated with the initial substrates of the pathway to be modified in order as they flow down the channel. This process would reduce unwanted side reactions, and greatly reduce the purification costs associated with producing biomolecules. The enzymes for the pathway would be slightly modified using an Avi-Tag so that they may be biotinylated by E. coli. These enzymes would be expressed in liquid bacterial cultures for each enzyme that also have been transformed with the light lysis plasmid. The cultures would then be exposed to the green light, and after the proper induction time, the culture would be pumped through the channel coated with streptavidin. The use of microfluidics is necessary to direct the flow of lysate to only the region designated for each enzyme. The benefit of using the light sensor system is the ability flow the cell culture through the channel as it lyses which minimizes the possible time for the enzymes to degrade. The simplicity of the lysis process allows for large bacterial cultures to be lysed with ease, allowing for the scalability, and automation of the construction process.
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Latest revision as of 17:08, 23 August 2011