"

From 2011.igem.org

Contents 1 Overall Project Description 2 Light Induced Lysis 3 Violacein Pathway 4 Microfluidic Device 5 DNA Assembly Methods

Overall Project Description

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.

Light Induced Lysis

Violacein Pathway

We chose to use the violacein pathway fully characterized by Balibar et. al.¹ as our model enzyme-mediated-reaction. The violacein pathway involves five enzymes, VioA, VioB, VioC, VioD, and VioE, in the conversion of L-Tryptophan into a violacein, purple chromophore. This pathway was an especially attractive candidate for our project not only because it has been thoroughly characterized in E coli., but also because relatively few enzymes are needed to convert a cheap, common substrate into a visualizable. Furthermore, only three of the five enzymes (VioA, VioB, and VioE) are required to produce a colored produce. Even with the exception of Vios C and D, L-Tryptophan is converted into prodeoxyviolacein, a green pigment (Figure 3A in Balibar et. al.¹). Thus, we chose to biotinylate only VioA, VioB, and VioE to provide proof-of-concept that enzymes binded to our microfluidic devices may be used to facilitate enzyme-mediated reactions.

Violacein Pathway Figure from Balibar et. al. ¹

Microfluidic Device

DNA Assembly Methods

1. In Vitro Biosynthesis of Violacein from l-Tryptophan by the Enzymes VioA−E from Chromobacterium violaceum† Carl J. Balibar and and Christopher T. Walsh Biochemistry 2006 45 (51), 15444-15457. http://pubs.acs.org/doi/abs/10.1021/bi061998z