iGEM Tokyo Tech 2011 Team

1. Cool down in summer with our Rock, Paper and Scissors Game

When summer comes it brings vacations, so students have plenty time to have fun. But summer means hot weather! At times we wish we could just make it rain to refresh ourselves. In light of these circumstances, and given the opportunity to do experiments with E. coli through our iGEM project activity, we designed a game that can be played between E.coli and humans. Still more, the winner of the game can get refreshing prizes! Dear students, let us introduce you to the first human-bacteria Rock, Paper and Scissors game! Win and get a rain shower, plus a “urea cooler”!

2. Rock, Paper and Scissors (RPS) game

So, how to play RPS with a handless bacteria? Instead of hands, we designed our game so that we could use different signaling molecules corresponding either to rock, paper or scissors. In our set of six signaling molecules, humans use IPTG, aTc and salicylate. E. coli uses 3O-C6-HSL, 3O-C12-HSL and AI-2. In each case, these signaling molecules correspond to rock, paper and scissors respectively.

The next step is to design a way to know who wins the RPS game. That is when the E. coli judge team comes in! Each judge has an AND-gate promoter which produces a single output based on two inputs, human side and E.coli side signaling molecules. Since we want the output to be visible, we use either GFP, RFP or CFP to indicate whether humans win, lose or it is a tie, respectively.

Lastly, we need to make sure E.coli follows the rules of the game by synthetizing only one signaling molecule every time it plays. Importantly, we have to design the opponent E.coli to be able to choose its signal randomly for a fare game, because E.coli will continue to lose if E.coli always produce same signaling molecule. In a view of these needs, we designed three randomizers that satisfy the conditions for the game. Single Colony Isolation, Survival of single strain and Conditional Knockout.

3. Make it rain

Playing RPS with E. coli during summer was fun, but, even if humans won, celebrations did not last long since we soon returned to complaining about the hot weather. As a prize for humans who win in our RPS game, we designed an E.coli that can make it rain, making the hot summer more fun and refreshing (let alone applications in agriculture). To make it rain we aimed to construct an isoprene synthetizing E.coli. Photo-oxidized isoprene acts as a condensation nucleus (Colin D. O‘Dowd et al., 2002, Nature) and can cause shower (make it rain) if only it is present in very low concentrations.

4. Urea cooler

Coolers can be made by adding urea to water, since dissolving urea in water is an endothermic reaction (-57.8 cal/g). By only making arginase production in E. coli, we can make the bacterium synthetizeing urea! Furthermore, we increased urea production by introduction of Arg Box sequence, which absorb ArgR repressor, at a site distant from promoters.
To analyze how we obtain even more urea from our E.coli, elementary flux analysis (Schuster 2000) identified metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. Making use of it we can tell which substrates and pathways are the best for obtaining more urea.