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From 2011.igem.org

Overall Project

Our project design essentially involves two parts. First, we would like to develop a genetic switch that is sensitive to specific wavelengths of visible light and use this gene expression system 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 specific gene expression. The system is composed of a light-activated surface protein, which autophosphorylates an intermediate chromophore, and a reporter protein that binds to a specific promoter and is activated by the chromophore.

The genes to be expressed downstream of the promoter make up a lysis cassette derived from the lambda phage and developed by Prof. Young at Texas A&M University. 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 lyse bacterial cultures within a known timeframe and with specificity to green light.

The second part of the project is an interesting application for the light-induced lysis system. We plan on binding the enzymes of a known biosynthetic pathway to the surface of a microfluidic channel and organizing them in a linear, chronological order. This would allow for a solution saturated with the initial substrate of the pathway to be modified in order as it flows down the channel. This process would reduce unwanted side reactions and greatly reduce the purification costs associated with producing biomolecules.

In extension to the work developed by the 2009 University of Cambridge iGEM team, our project features the synthetic pathway for violacein. This purple pigment can be found in nature as the compound responsible for giving Chromobacterium violaceum its metallic violet sheen. Studies suggest that violacein is also significant for its anti-bacterial and cancer-fighting properties. While the product in its fully mature, purple-colored form requires five enzymes, we focus on a dark green precursor, prodeoxyviolacein, that only requires three of the five: VioA, VioB, and VioE (a catalyst).

Binding these three enzymes to the surface of the microfluidics device takes advantage of the strong affinity between biotin and streptavidin. While the channel is coated with streptavidin, the enzymes of the synthetic pathway are modified to include an Avi-Tag for biotinylation by E. coli. VioA, VioB, and VioE are individually expressed in separate liquid cultures of E. coli that have been transformed with the plasmid coding for the light-inducible lysis kit. The cultures would then be exposed to the appropriate green light. After the proper induction time, biotin-tagged enzyme is released into the resulting cell lysate, which is then 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 with a known time frame is the ability to flow the cell culture through the channel as soon as lysis and release of enzyme begins. This minimizes the possible time for the newly synthesized enzymes to degrade in the harsh environment of bacterial lysate. The simplicity of the lysis process allows for large bacterial cultures to be ruptured with ease, allowing for the scalability and automation of the construction process.

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.