Here's how you can play Colisweeper!

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:

a non-mine colony,

a non-mine colony located close a mine, when played will reveal the number of surrounding mines , which on addition of a multi-substrate reveals the number of mines in the vicinity of this colony

a dangerous and scary 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 this mine after which this colony cannot be played again and the game is continued.

The biology behind the game

Figure 2. Signal transduction from secreted messenger to colorimetric response.

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.

The biology is explained here. The sender colony secretes the quorum signal 3-oxo-N-hexanoyl-L-homoserine lactone (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.

The Game Board

Figure 2. The game board pattern

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 (OHHL) and therefore they must be placed in such a manner that equal amount of signal is received in adjacent non-mine colonies.

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.
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. 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.

What about signaling and information processing ?

Signal expression by mine cells (sender cells)

Mine colonies constitutively express the "mine-cell reporter” that reacts with the multi-substrate to produce a colored product that indicates a mine colony. Addition of the multi-substrate turns the colony black, indicating the mine and hence the game ends.
At the same time, the mine colony is a sender module of OHHL. LuxI preceded by a constitutive promoter, which synthesizes OHHL signalling molecules. This means that the mine continuously produces OHHL, allowing it to gradually diffuse radially out to neighboring colonies. Thus the mine colonies sends out the signalling molecule OHHL that is processed by the receiver cells via diffusion in the agar mine grid .

Information processing by non-mine cells (receiver cells)

Figure 3. [OHHL] dependent information processing

So what exactly happens with the expressed OHHL?

1. Receiver cells respond to low OHHL concentration 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.

2. Receiver cells respond to high OHHL concentration 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.

3. Receiver cells signal no mines in their vicinity 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.

Flagging

Remember what the flagging does in a traditional minesweeper game? It helps us put a red flag on the dangerous spots without detonation!

If the player is sure about one colony to be a mine he has the option to flag it as a visual help. E.coli expressing constitutively express a reporter, which reacts with substrate #2 and changes the colony’s color to green.

Remember! Flagging doesn’t reveal any information about mines in neighbouring cells.