Team:ETH Zurich/Project

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<h1>Here's how you can play Colisweeper!</h1>
 
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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">Gaining inspiration from the computer game Minesweeper, we aspire to incorporate the same rules in the biological version as well. To replicate the "mouse click"  in the computer game, you can add a substrate to the colony of your choice that leads to an enzyme-susbtrate reaction that expresses the reporter.The agar mine field consists of colonies which can be one of the following:<br><br><b>a non-mine colony</b>,<br><br><b> a non-mine colony located close a mine, when played will reveal the number of surrounding mines  </b>, which on addition of a multi-substrate reveals the number of mines in the vicinity of this colony <br><br><b> a dangerous and scary mine colony</b>,which when played reveals the mine color and the game is over<br><br> You also have the option to flag a mine - if you are certain that a colony is a mine, you can flag this mine after which this colony cannot be played again and the game is continued.</p>
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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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<br><br>
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<h1> Colisweeper work?</h1>
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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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[[File:Infoprofinal.png|500px|left|thumb|<b>Figure 2. Signal transduction from secreted messenger to colorimetric response.</b>]]
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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 which are the sender cells and 2. the non-mine strain which are 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 click and the right click of the mouse is simulated with the addition of the multi-substrate mix and the single substrate.<br><br>
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The biology is explained here. The sender colony secretes the quorum signal 3-oxo-N-hexanoyl-L-homoserine lactone ([https://2013.igem.org/Team:ETH_Zurich/Experiments_2 OHHL]) that diffuses through the agar to the surrounding cells.The receiver cells receive the OHHL and convert the inactive luxR to active luxR by forming a complex OHHL-luxR. The information is translated via mutated pLuxR promoters which have different OHHL affinities, after processing which leads to the secretion of different hydrolases. The player can see by eye ,and in less than 5 minutes after the addition of the substrates, the number of mine colonies surrounding the played colony.</p>
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<h1>The Game Board</h1>
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[[Image:Hexstructure.png|300px|right|thumb|<b>Figure 2. The game board pattern</b>]]
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<p align="justify">The design of the mine field was not so intuitive. The traditional minesweeper pattern is a grid with tightly spaced squares, where each square has 8 different adjacent squares around it. To replicate such a grid in the biological game would be a complex one. Moreover, our mine colonies sends the OHHL signal through the diffusion in the agar ([https://2013.igem.org/Team:ETH_Zurich/Experiments_2 OHHL]) and therefore they must be placed in such a manner that equal amount of signal is received in adjacent non-mine colonies.  
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In the traditional minesweeper pattern, if we located bacterial colonies in the place of squares, the colonies placed in the corners will be further away compared to the colonies along the edges.  
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We came up with the hexagonal honeycomb-like pattern where colonies are placed at all edges of the hexagon except from the center. In this manner, colonies are plated in a hexagonal pattern throughout the agar plate and so we have the number of mine colonies restricted to one, two or three mines that can surround a non-mine colony.  
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To plate the field liquid cultures of mine and non-mine cells are grown to an OD600 of 0.5, then, using a pipette, 1.5ul of liquid culture are placed according to the grid.
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<h1>What about signaling and information processing ?</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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<b>Signal expression by mine cells (sender cells)</b>
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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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<p>Mine cells are a strain of <i>E.coli</i> bacteria that constitutively express the "mine-cell reporter” reacting with substrate #1 to produce the visual effect of being a mine. After substrate #1 is added the bacterial colony turns black, indicating our defeat.<br>At the same time, the mine cell is a sender module of OHHL. It has the LuxI preceded by a constitutive promoter, which induces OHHL expression. This means that the mine continuously produces OHHL, allowing it to gradually diffuse radially out to neighboring colonies. We use this attribute of our mine cells for quorum sensing in the surrounding bacterial cells.</p>
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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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<b>Information processing by non-mine cells (receiver cells) </b>
 
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[[File:Circuit2 colisweeper.png|600px|left|thumb|<b>Figure 3. [OHHL] dependent information processing</b>]]
 
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So what exactly happens with the expressed OHHL?
 
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<p align="justify">1. Receiver cells respond to low OHHL concentration
 
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If one mine cell is close to the colony which is selected with substrate #1, the OHHL concentration induces a certain reaction. Thanks to a bandpass filter using the LuxR system, the cell is able to detect low concentrations of OHHL and activate a reporter signalling low OHHL concentration. The mine changes color to blue and the player (that’s you!) knows that one mine is close to the cell he had just revealed.
 
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2. Receiver cells respond to high OHHL concentration
 
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Now say there are two or more mine cells directly next to our chosen colony. There is therefore a proportionally higher concentration of diffused OHHL in the selected cell. The LuxR system works as a bandpass filter again and induces the expression of another reporter to show that 2 or more mines are surrounding the selected colony. The field changes color to green to signal that to the player. Ideally, thanks to the colors,  the player is always sure how many mines surround an uncovered field.
 
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<br><br>3. Receiver cells signal no mines in their vicinity
 
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If there are no mine cells directly next to a colony, it will not have any OHHL. The LuxR bandpass system will respond and the bacteria will express a reporter indicating that this is a "neutral" cell.</p>
 
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<h1>Flagging</h1>
 
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<p>Remember what the flagging does in a traditional minesweeper game? It helps us put a red flag on the dangerous spots without detonation!
 
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If the player is sure about one colony to be a mine he has the option to flag it as a visual help. <i>E.coli</i> expressing constitutively express a reporter, which reacts with substrate #2 and changes the colony’s color to green.
 
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Remember! Flagging doesn’t reveal any information about mines in neighbouring cells.</p>
 
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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.


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