Team:ETH Zurich

From 2013.igem.org

(Difference between revisions)
Line 31: Line 31:
</li>
</li>
<li><b><br>Hydrolase Reactions</b><br><br> As a reporter system we use a set of orthogonal hydrolase enzymes: alkaline phosphatase (<i>phoA</i>), β-galactosidase (<i>lacZ</i>), acetylesterase (<i>aes</i>), β-N-Acetylglucosaminidase (<i>nagZ</i>) and β-glucuronidase (<i>gusA</i>). Each hydrolase can react with its respective substrate within minutes resulting in a fast, colorful output. Quick response times and the ability to read the output without using instruments are essentials for a fast gameplay.</li>
<li><b><br>Hydrolase Reactions</b><br><br> As a reporter system we use a set of orthogonal hydrolase enzymes: alkaline phosphatase (<i>phoA</i>), β-galactosidase (<i>lacZ</i>), acetylesterase (<i>aes</i>), β-N-Acetylglucosaminidase (<i>nagZ</i>) and β-glucuronidase (<i>gusA</i>). Each hydrolase can react with its respective substrate within minutes resulting in a fast, colorful output. Quick response times and the ability to read the output without using instruments are essentials for a fast gameplay.</li>
-
<li><b><br>The Model</b><br><br>As our bio-game is based on processing the AHL concentration in the non-mine colonies, the diffusion of AHL 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 AHL 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). Our model turned out to be very valuable in the circuit refinement and the design of experiments. Moreover, we continually improve out model by incorporating parameters from our own experimental data.
+
 
 +
<li><b><br>The Model</b><br><br>As our bio-game is based on processing the AHL concentration in the non-mine colonies, the diffusion of AHL in the agar is vital to the system. The diffusion was modeled by carrying out simulations to determine the time and distance of diffusion. We also 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). Our model turned out to be very valuable in the circuit refinement and the design of experiments. Moreover, we continually improve out model by incorporating parameters from our own experimental data.
</li>
</li>
<li><b><br>Experimental Results</b><br><br> We performed a lot of diffusion experiments in order to determine the distance between colonies in the grid, the incubation time and the strengh of the promoter used to activate the LuxI. The dialog with the model was very strong in this part. We set-up a proof-of-principle  using GFP as reporter system. The LuxR promoter from registry has to be mutated to obtain a library of LuxR promoter with different sensitivities in order to distinguish between different levels of AHL. After the first tests with the final reporter system : the hydrolases, we need to review the circuit to reduce the leakiness of the Plac promoter responsible for the LuxR activation. We came up with a glucose shutdown of the Plac promoter and a negative feedback loop using lacI. Meanwhile we characterize the biobricks using various methods like Michealis-Menten kinetics and flow cytometry.
<li><b><br>Experimental Results</b><br><br> We performed a lot of diffusion experiments in order to determine the distance between colonies in the grid, the incubation time and the strengh of the promoter used to activate the LuxI. The dialog with the model was very strong in this part. We set-up a proof-of-principle  using GFP as reporter system. The LuxR promoter from registry has to be mutated to obtain a library of LuxR promoter with different sensitivities in order to distinguish between different levels of AHL. After the first tests with the final reporter system : the hydrolases, we need to review the circuit to reduce the leakiness of the Plac promoter responsible for the LuxR activation. We came up with a glucose shutdown of the Plac promoter and a negative feedback loop using lacI. Meanwhile we characterize the biobricks using various methods like Michealis-Menten kinetics and flow cytometry.

Revision as of 21:11, 26 October 2013

Header2.png
80px-Eth igem logo.png

Minesweeperheader.png

Gold-medal.png
Play Minesweeper here !

  • Colisweeper
    Colisweeper is an interactive, biological version of the computer game Minesweeper. The goal is to clear an agar “minefield” without detonating the mines. Genetically engineered Escherichia coli colonies are used as mines and non-mines. Mines secrete the signaling molecule AHL whereas non-mines process the signal. To distinguish different AHL-levels, a library of PLuxR promoters with various AHL 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.

    The slideshow covers all aspects of our project. Feel free to CLICK ON ANY PICTURE in ANY SLIDESHOW to navigate to the description page.

    CLICK ON THE START BUTTON OF THE FIRST SLIDE TO SEE OUR VIDEO

  • The computer game Minesweeper

    The computer game consists of a square lattice grid with hidden squares that can be mines or non-mines. The goal of the game is to clear the field without detonating the mines. The identity of a square is revealed by left clicking on it: the digit revealed represents the number of adjacent mines. The player uses this information to decide which square can be a potential mine and flags it. A square can be flagged by a right click. The game is won when all the mines and only the mines are flagged. If a mine is encountered, it is detonated along with all other mines and the game is over.

  • Information Processing

    Sender cells (mines) express LuxI protein, which catalyzes the production of a small signalling molecule, called AHL. This molecule diffuses through the agar plate and reaches the receiver cells called non-mine colonies. These colonies are designed to distinguish different concentrations of AHL and translate this analog information into the expression of different sets of reporter enzymes. The continuous signal is digitized through a set of mutated PLuxR promoters with different AHL sensitivities acting as highpass filters.

  • From Minesweeper to Colisweeper

    Mines secrete the signaling molecule AHL which diffuses through the agar and is processed by neighboring non-mine colonies. High-pass filters were constructed to control the expression of different orthogonal hydrolase enzymes in non-mines. Promoters that serve as high-pass filters were tuned to express hydrolases depending on the concentration of the AHL 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 genomic expression of lacZ enables the flagging of both mines and non mine colonies turning the colonies green.

  • Hydrolase Reactions

    As a reporter system we use a set of orthogonal hydrolase enzymes: alkaline phosphatase (phoA), β-galactosidase (lacZ), acetylesterase (aes), β-N-Acetylglucosaminidase (nagZ) and β-glucuronidase (gusA). Each hydrolase can react with its respective substrate within minutes resulting in a fast, colorful output. Quick response times and the ability to read the output without using instruments are essentials for a fast gameplay.

  • The Model

    As our bio-game is based on processing the AHL concentration in the non-mine colonies, the diffusion of AHL in the agar is vital to the system. The diffusion was modeled by carrying out simulations to determine the time and distance of diffusion. We also 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). Our model turned out to be very valuable in the circuit refinement and the design of experiments. Moreover, we continually improve out model by incorporating parameters from our own experimental data.

  • Experimental Results

    We performed a lot of diffusion experiments in order to determine the distance between colonies in the grid, the incubation time and the strengh of the promoter used to activate the LuxI. The dialog with the model was very strong in this part. We set-up a proof-of-principle using GFP as reporter system. The LuxR promoter from registry has to be mutated to obtain a library of LuxR promoter with different sensitivities in order to distinguish between different levels of AHL. After the first tests with the final reporter system : the hydrolases, we need to review the circuit to reduce the leakiness of the Plac promoter responsible for the LuxR activation. We came up with a glucose shutdown of the Plac promoter and a negative feedback loop using lacI. Meanwhile we characterize the biobricks using various methods like Michealis-Menten kinetics and flow cytometry.

  • 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!

Retrieved from "http://2013.igem.org/Team:ETH_Zurich"