Information processing and project overview

Our game Colisweeper is played on an agar mine-field, which is a petri dish with E.coli colonies. Some of these colonies are mines and others non-mines. The mines serve as the sender cells and the non-mines serve as the receiver cells.

Signal origin: The sender cells produce the signaling molecule 3-oxo-N-hexanoyl-L-homoserine lactone (AHL) by constitutive LuxI production, which diffuses freely in and out of cells. This signal serves as the means of communication via quorum sensing between mine colonies and non-mine colonies.

Pre-Processing: The agar minefield consists of mine and non-mine colonies in a honey-comb grid. The colonies are placed on the edges of each hexagon. This way, each colony is restricted to three neighboring colonies. The senders produce the signalling molecule AHL that diffuses through the agar. Depending on the amount of diffused AHL that is processed in the non-mines, differentially expressed proteins indicate the number of mines; 0 mines, 1 mine and 2 mines around (to find out more about the grid pattern, please click here). In the receiver cells, the signaling molecule forms a complex with the inactive LuxR to form an active complex AHL-LuxR.

Processing: Diffused AHL molecules from the sender cells are processed in the non-mines via complex formation at subsequential binding to P_LuxR promoters with different AHL sensitivities. The interaction of the AHL with the AHL-sensitive promoters results in expression of different enzymes: orthogonal hydrolases .

Optimization: Proof-of-principle experiments with sender-receiver set-up and GFP as the reporter suggested leakiness in our system. To reduce the leakiness, we optimized our system (Please see the optimization part for more details).

Player interaction : To play the game, the player pipettes a substrate-mix on a colony, which leads to a color change of the colony. This gives the player information to logically carry out the next move in the game. The left click in the computer game corresponds to pipetting a multi-substrate mix on the colony in the bio-game. To mimic the flagging option of right clicking in the bio-game, the player can flag a colony by adding either a flagging solution that turns a colony into green color or by adding the Remazol blue dye. If the dye is added on to a flagged colony, the player can unflag the colony again by adding an enzyme laccase that removes the color. For more details please click here

Output: Addition of the playing solution (multi-substrate mix) or flagging solution(single substrate) gives color within minutes due to specific conversion of the substrates by the hydrolases which indicates the identity of the played colony. The color output is based on an overlay of different expressed hydrolases in the different situations.

Figure 1:Information processing from secreted signaling molecule to colorimetric response. The signal diffuses through the agar from sender cells (light blue) to receiver cells (dark blue). The non-mine colonies are designed to distinguish between different concentrations of AHL and translate this information into expression of different hydrolases. The expression is driven by different P_LuxR promoters that show different AHL sensitivities and serve as high pass filters. After an incubation time of 12 hours the player pipettes a substrate on the colony. The hydrolase converts the substrate into a colored product which is visible by eye.

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