Team:ETH Zurich

From 2013.igem.org

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<li><b><br>From Minesweeper to Colisweeper </b><br><br> Mines secrete the signaling molecule OHHL whereas non-mines process the signal after diffusion of OHHL through the agar. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines. These were tuned to express hydrolases depending on the concentration of the OHHL molecules from the surrounding mines. The colors yellow, salmon and magenta corresponds to zero, one and two mines around a colony. Additionally, the mines express their own hydrolase which when added with the multi-substrate gives blue color. The constant expression of ''lacZ'' enables the flagging of both mines and non mine colonies turning the colonies green.  
<li><b><br>From Minesweeper to Colisweeper </b><br><br> Mines secrete the signaling molecule OHHL whereas non-mines process the signal after diffusion of OHHL through the agar. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines. These were tuned to express hydrolases depending on the concentration of the OHHL molecules from the surrounding mines. The colors yellow, salmon and magenta corresponds to zero, one and two mines around a colony. Additionally, the mines express their own hydrolase which when added with the multi-substrate gives blue color. The constant expression of ''lacZ'' enables the flagging of both mines and non mine colonies turning the colonies green.  
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<li><b><br>The Model</b><br><br>As our system is based on sensing the OHHL concentration, the diffusion of the signalling molecule in the mine field is a vital part of the model; we carried out simulations to determine time and distance scales. In addition to OHHL diffusion, we modelled synthesis, regulation and degradation reactions of the molecules involved in our genetic circuits. To account for both processes: diffusion and reactions, we developed a spatio-temporal model in two dimensions comprised by three modules: mines, receivers, and the agar plate. To solve the system of partial differential equations (PDEs) we used finite element methods. The information from the model was used to validate and improve our system.  
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<li><b><br>The Model</b><br><br>As our bio-game is based on processing the OHHL concentration in the non-mine colonies, the diffusion of OHHL in the agar is vital to the system. The diffusion was modeled by carrying out simulations to determine the time and distance of diffusion. In addition to OHHL diffusion, we modeled synthesis, regulation and degradation reactions of the proteins involved in our genetic circuits. To account for both processes: diffusion and reactions; we developed a spatio-temporal model in two dimensions comprised by three modules: mines, receivers, and the agar plate. Finite element methods were used to solve the system of partial differential equations (PDEs). The information from the model was used to validate and improve our experimental system.  
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<li><b><br>High pass filter</b><br><br>To distinguish between OHHL-levels, a library of pLuxR promoters with various OHHL sensitivities was created through site-saturation mutagenesis. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines, depending on the number of surrounding mines.
<li><b><br>High pass filter</b><br><br>To distinguish between OHHL-levels, a library of pLuxR promoters with various OHHL sensitivities was created through site-saturation mutagenesis. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines, depending on the number of surrounding mines.

Revision as of 23:23, 4 October 2013

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  • Colisweeper

    Colisweeper is an interactive, biological version of the Minesweeper computer game, based on quorum sensing and chromogenic enzymatic reactions. The goal is to clear an agar “minefield” without detonating the mines. Genetically engineered Escherichia coli colonies are used as sender-cells (mines) and receiver-cells (non-mines). Mines secrete the signaling molecule OHHL (analog of AHL: N-acyl homoserine lactone) whereas non-mines process the signal. To distinguish between OHHL-levels, a library of PLuxR promoters with various OHHL sensitivities was created through site-saturation mutagenesis. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines, depending on the number of surrounding mines. Additionally, the mines express their own hydrolase.

  • Colisweeper video

    Check out the animation of our bio-game Colisweeper !

  • The computer game Minesweeper

    The computer game is played on a square lattice mine field with mines and non-mines. The goal of the game is to clear the field without detonating the mines. By left clicking on each square, the identity of the square is revealed. The reveled digits 1, 2, or 3 represent that there are one, two or three mines adjacent to the clicked square. Empty squares have no mines adjacent to them. The player uses this logic to decide which squares are mines and flags it. The mines can be flagged by right clicking on the square. The game is won when all the mines are flagged in the mine field. If a mine is clicked, all the other mines in the mine field are detonated and the game is over.

  • Gameplay

    The game Colisweeper is played on an agar petridish with the mine and non-mine colonies plated in a hexagonal lattice mine field. The player has to pipette colorless substrates on a colony. A single move of pipetting would require the player to choose between two colorless substrates. If the multi-susbtrate is added, this reveals the identity of the colony- as in the number of mines surrounding a non-mine. The single colorless substrate is pipetted onto a colony if the player is certain of a mine colony. Addition of either substrates produces a defined colored product within minutes, allowing identification of the played colony and the number of mines surrounding it.

  • Information Processing

    The non-mine colonies express the reporters that are induced by OHHL. The OHHL is produced by the mine colonies. The reporters are to be expressed under different concentrations of OHHL. In order to distinguish between different concentrations of OHHL and translate this information into expression of different sets of reporters, the non-mine colonies are equipped with mutated PLuxR promoters with different OHHL sensitivities. This serves as high-pass filters for the reporters. The mutated promoters were created using site-saturation mutagenesis. Through mutation of the LuxR protein binding sites we were able to tune the promoters to different OHHL affinities.

  • Hydrolase Reactions

    We use a set of orthogonal hydrolases as our reporter system. The player adds a multi-substrate to the colony and the hydrolase reacts within minutes to produce a visible color. Each color represents the proximity of the mines close to the played colony. The set of hydrolases are alkaline phosphatase (phoA), β-galactosidase (lacZ), acetylesterase (aes), β-N-Acetylglucosaminidase (nagZ) and β-glucuronidase (gusA). They react with the specific substrates in the multi-substrate to achieve fast and colorful outputs with each color indicative of the next logical move for the player.

  • From Minesweeper to Colisweeper

    Mines secrete the signaling molecule OHHL whereas non-mines process the signal after diffusion of OHHL through the agar. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines. These were tuned to express hydrolases depending on the concentration of the OHHL molecules from the surrounding mines. The colors yellow, salmon and magenta corresponds to zero, one and two mines around a colony. Additionally, the mines express their own hydrolase which when added with the multi-substrate gives blue color. The constant expression of ''lacZ'' enables the flagging of both mines and non mine colonies turning the colonies green.

  • The Model

    As our bio-game is based on processing the OHHL concentration in the non-mine colonies, the diffusion of OHHL in the agar is vital to the system. The diffusion was modeled by carrying out simulations to determine the time and distance of diffusion. In addition to OHHL diffusion, we modeled synthesis, regulation and degradation reactions of the proteins involved in our genetic circuits. To account for both processes: diffusion and reactions; we developed a spatio-temporal model in two dimensions comprised by three modules: mines, receivers, and the agar plate. Finite element methods were used to solve the system of partial differential equations (PDEs). The information from the model was used to validate and improve our experimental system.

  • High pass filter

    To distinguish between OHHL-levels, a library of pLuxR promoters with various OHHL sensitivities was created through site-saturation mutagenesis. High-pass filters were constructed to control the expression of different orthogonal hydrolases in non-mines, depending on the number of surrounding mines.

  • Human practice

    Inspired by our Colisweeper project, we analyzed the relationship between synthetic biology and games. For one thing synthetic biology can be used to play common games in a new way, possibly for educational purposes or as a basis for proof-of-principle experiments for new circuits. More recently synthetic biologists also started to use games as a research tool, an innovative approach to make use of crowd-sourcing and distributed computing. We want to find correlations and discuss possible consequences for Synthetic Biology.

  • Team

    We are a team of seven highly motivated Bachelor- and Master Students at ETH Zürich pursuing various fields such as Biotechnology, Biomedical Engineering, Neurobiology and Bioinformatics. The iGEM project is carried out at one of the youngest departments of ETHZ located in Basel-Department of Biosystems Science and Engineering - flourishing in interdisciplinary biological research. If you're around Basel, make sure to visit our team's lab to play the bio-game Colisweeper!

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