Team:ETH Zurich/Project

From 2013.igem.org

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<h1>Information processing</h1>
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<p align="justify">Each minesweeper square in the computer game corresponds to a bacterial colony on the agar mine-field. We have two bacterial strains: 1. the mine strain with the sender cells and 2. the non-mine strain with the receiver cells. The cells communicate through diffusion of OHHL and change color to give the player information in order to logically carry out the next move in the game. The colonies remain white as seen on the agar plate until any substrate is added. The left and the right click of the mouse is simulated with the addition of the multi-substrate mix and the single substrate.</p>[[File:infoproc14.png|800px|center|thumb|<b>Figure 1: Signal transduction from secreted signaling molecule to colorimetric response.</b>]]
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<p align="justify">The biology is explained here. The sender colony secretes the quorum sensing molecule 3-oxo-N-hexanoyl-L-homoserine lactone
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([https://2013.igem.org/Team:ETH_Zurich/Experiments_2 OHHL]) that diffuses through the agar to the surrounding cells. The receiver cells passively take up the OHHL by diffusion where the signaling molecule forms a complex with the inactive LuxR to activate it. The information is translated via mutated pLuxR promoters of different OHHL affinities which leads to the secretion of different hydrolases. Within minutes after the addition of substrate a change in color indicates the identity of the played colony and number of surrounding  mine colonies.
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If we located bacterial colonies in the place of squares like in the traditional game, the colonies placed in the corners would be further away compared to the colonies along the edges. We decided on a hexagonal honeycomb-like pattern, where colonies are placed at all edges of the hexagon except from the center. In this set-up the number of mine colonies restricted is to only one, two or three that can surround a non-mine colony. This also facilitates the selection of suitable reporter enzymes. To plate the field, liquid cultures of mine and non-mine cells are grown to an OD<sub>600</sub> of 0.5, then, using a pipette, 1.5μl of liquid culture are placed according to the grid.
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<h1><b>How to play minesweeper using E.Coli</b></h1>
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<h1>How is Colisweeper played ?</h1>
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[[File:Project description10.png|300px|left|thumb|<b>Figure 2: Project description</b>]]
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<div style="position:absolute;left:1150px;top:640px">[[Image:Minesweeperexample.jpg|170px]]</div>
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<p align="justify">Inspired by the computer game Minesweeper, we aspire to incorporate all the same features and rules in the biological version. To replicate the "mouse click" in the computer game, you can add a substrate to the colony of your choice which leads to a colorful enzyme-susbtrate reaction. The agar mine field consists of colonies which can be one of the following:
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<br><br><b>a non-mine colony</b>, which will display the "safe colour" when played
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<div style="position:absolute;top:270px;left:640px;width:50%;height:auto;font-family:Comic Sans MS;font-size:20px;color:white">As you certainly know a classic minesweeper has 4 options. Non mine cells opening without any event, fields next to a mine indicating how many mines they are close to (1,2,3,4...), mine cells itself ending the game until one is revealed and last but not least the flagging option to mark fields as mine fields.<br><br>The goal of the game is to reveal all non mines as fast as possible.<br><br>Actually to simulate the "click" we decided to add a substrate starting a reaction or expressing a reporter.To play this with E.coli we need first of all non mine cells expressing a neutral reporter. We also choose to use quorum sensing for the bacteria close to a mine cell to know about how many mines are close to our cell. For the mine cell we will use a second colony expressing a different reporter obviously. The non mine cells and mine neighbouring cells are the same colony an the expression of the different reporters will be regulated by a band-pass filter for quorum sensing<br>To play in a comfortable way the substrates will be mixed in one solution.<br>Finally for the flagging option a constitutive expressed reporter is used with a separate substrate.<br><br> Now you're ready to sweep baby !<br>But let's have a closer look...</div>
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<br><b>a non-mine colony located close to one or more mines</b>, which on addition of a multi-substrate reveals the number of mines in the vicinity of this colony  
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<br><b>a mine colony</b>, which when played reveals the mine color and the game is over
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<br><br>You also have the option to flag a mine - if you are certain that a colony is a mine, you can flag it using a second substrate.</p>
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<div style="position:relative;top:align;padding:10px"><h1><b>The set up</b></h1></div>
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<div style="position:absolute;top:810px;padding:10px;width:50%;height:auto;font-size:20px;font-family:Comic Sans MS;color:white">The simplest set up is to place E.coli colonies on an M9 agar plate in a certain pattern.<br><br> But how to choose the right pattern ?<br><br>The more intuitive way is to use the classic minesweeper pattern with squares, plating the colonies in the corner of the sqaures. The problem is we are working with the diffusion of <i>Acylated homoserine lactones (AHL)</i> therefore the colonies must be at the same distance. But in this pattern if we consider one colony X, 4 colonies will be at a distance 1, and 4 colonies will be at a distance &radic;2.We would not be able to simulate correctly the indication of mines around.<br><br>Finally the hexagonal honeycomb like pattern seems to be perfectly adapted for our use because every intersection and centre of the hexagons are at the same distance to the colonies around them.</div>
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<div style="position:relative;padding:10px"><h1><b>Mine cells / Sender cells</b></h1></div>
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<div style="position:absolute;top:1320px;left:590px;padding:10px;color:white;font-size:20px;font-family:Comic Sans MS">On the one hand the mine cells are constitutively expressing the "mine cell" reporter reacting with the substrate to the visible result of being a mine.<br><br>On the ohter hand the mine cell is a sender module of <i>Acylated homoserine lactones</i> for quorum sensing of the neighbouring colonies.<br> The LuxI preceded by a constitutive promoter induces AHL expression.<br><br> AHL expression level and diffusion has to be characterized during independent experiments to know about the diffusion speed and time to wait before playing.<br><br>Let's have a look what happens with the expressed AHL.</div>
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<div style="position:relative;padding:10px"><h1><b>Non mine cells neighbouring mine cells</b></h1></div>
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<div style="position:relative;right:-800px;padding:10px">[[Image:Lowmineconc.png|400px]]</div>
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<div style="position:absolute;top:1680px;padding:10px;width:50%;height:auto;font-size:20px;font-family:Comic Sans MS;color:white">Receiver cell response to low AHL concentration<br><br>If one mine cell is close to the colony which is selected with substrate, the AHL concentration induces different reporter possibilities.Thanks to a band pass filter using the LuxR system the cell is able to detect low concentrations of AHL and activate a reporter signalling low AHL concentration. <br><br>The Gamer knows that 1 mine is close to the mine he just revealed.</div>
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<div style="position:relative;right:-700px;padding:15px">[[Image:highmineconc.png|500px]]</div>
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<div style="position:absolute;top:1940px;padding:15px;width:50%;height:auto;font-size:20px;font-family:Comic Sans MS;color:white">Receiver cell response to high AHL concentration<br><br>Consider two or more mine cells producing AHL surround the selected colony, the AHL concentration is higher, the LuxR system working as band pass filter induces the expression of an other reporter to to show that 2 or more mines are surrounding the selected colony.<br><br>The goal to achieve is that the player exactly knows how many mines are in the surrounding.</div>
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<h1><b>Experiments</b></h1>
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<h1><b>Results</b></h1>
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Latest revision as of 10:05, 3 October 2013

Header2.png
80px-Eth igem logo.png

Information processing

Each minesweeper square in the computer game corresponds to a bacterial colony on the agar mine-field. We have two bacterial strains: 1. the mine strain with the sender cells and 2. the non-mine strain with the receiver cells. The cells communicate through diffusion of OHHL and change color to give the player information in order to logically carry out the next move in the game. The colonies remain white as seen on the agar plate until any substrate is added. The left and the right click of the mouse is simulated with the addition of the multi-substrate mix and the single substrate.

Figure 1: Signal transduction from secreted signaling molecule to colorimetric response.


The biology is explained here. The sender colony secretes the quorum sensing molecule 3-oxo-N-hexanoyl-L-homoserine lactone (OHHL) that diffuses through the agar to the surrounding cells. The receiver cells passively take up the OHHL by diffusion where the signaling molecule forms a complex with the inactive LuxR to activate it. The information is translated via mutated pLuxR promoters of different OHHL affinities which leads to the secretion of different hydrolases. Within minutes after the addition of substrate a change in color indicates the identity of the played colony and number of surrounding mine colonies.

If we located bacterial colonies in the place of squares like in the traditional game, the colonies placed in the corners would be further away compared to the colonies along the edges. We decided on a hexagonal honeycomb-like pattern, where colonies are placed at all edges of the hexagon except from the center. In this set-up the number of mine colonies restricted is to only one, two or three that can surround a non-mine colony. This also facilitates the selection of suitable reporter enzymes. To plate the field, liquid cultures of mine and non-mine cells are grown to an OD600 of 0.5, then, using a pipette, 1.5μl of liquid culture are placed according to the grid.


How is Colisweeper played ?

Figure 2: Project description

Inspired by the computer game Minesweeper, we aspire to incorporate all the same features and rules in the biological version. To replicate the "mouse click" in the computer game, you can add a substrate to the colony of your choice which leads to a colorful enzyme-susbtrate reaction. The agar mine field consists of colonies which can be one of the following:

a non-mine colony, which will display the "safe colour" when played
a non-mine colony located close to one or more mines, which on addition of a multi-substrate reveals the number of mines in the vicinity of this colony
a mine colony, which when played reveals the mine color and the game is over

You also have the option to flag a mine - if you are certain that a colony is a mine, you can flag it using a second substrate.