Signal production by mine cells (sender cells)

Mine colonies constitutively express the "mine-cell reporter” hydrolase nagZ that reacts with the specific substrate in the multi-substrate solution to produce a chromogenic product. The colony turns blue, indicating that the colony is a mine and hence the game ends. At the same time, the mine colony is a sender module of AHL. LuxI is expressed constitutively and synthesizes the AHL signaling molecule. This means that the mine continuously produces AHL, that radially diffuses out to neighboring colonies. The signaling molecule AHL is processed by the receiver cells (also see below).

Information processing in non-mine cells (receiver cells)
Processing the AHL

1. Receiver cells respond to low AHL levels: If one mine cell is close to a non-mine colony and AHL diffusion takes place to reach the non-mine colony. Due to a high-pass filter using the LuxR system in the non-mine colony, low concentrations of AHL are detected and activate a reporter. Addition of the multi-substrate to this colony produces a salmon color, which indicates one mine is adjacent to the colony just played.

The wild type promoter [http://parts.igem.org/Part:BBa_R0062 BBa_R0062 PLuxR] is used for the detection of low AHL concentrations and therefore becomes activated when one mine cell is in the vicinity of the receiver cell. Then the wild type promoter induces the expression of gusA, a hydrolase able to convert a specific substrate to a visible red output.

2. Receiver cells respond to high AHL levels: If there are two or more mine colonies adjacent to a non-mine colony, a higher concentration of diffused AHL will be processed by the receiver colony. The LuxR system works as a high-pass filter again and induces the expression of another reporter to show that two or more mines surround the selected colony. Addition of the multi-substrate to this colony produces a magenta color which indicates that more than one mine is adjacent to this colony.

The mutated promoter [http://parts.igem.org/Part:BBa_K1216007?title=Part:BBa_K1216007 PluxR BBa_K1216007] will be used in our final set-up to only respond to high concentrations of AHL and therefore be activated, when two sender cells are surrounding the receiver cell, and expressing the aes hydrolase responsible for the colorimetric conversion of the specific substrate to a visible purple output.

3. Receiver cells signal with no mines in their vicinity. If there is no mine adjacent to a non-mine colony, no AHL is processed. Only the constitutive hydrolase phoA is expressed, which upon addition of the multi-substrate will produce a chromogenic compound. This indicates that there are no mines adjacent to this colony and the game can be continued.

Flagging
Placing a flag on a mine prevents the mine from detonating. We have implemented this feature of the computer game in Colisweeper. If the player is sure about a colony to be a mine, a second single substrate can be added on to this colony. All colonies express the native hydrolase lacZ reporter for flagging, a green colored product indicates a flagged colony. But remember, flagging does not reveal any information about mines in neighboring cells.