1. Designing and constructing the cooperator and cheater
We have looked the previous iGEM projects and articles on the game theory. We decided to express GFP for cheater and beta-glucosidase for cooperator. We received the designed constitutive expression vector applicable to ligation independent cloning using the plasmid containing BBa_J23106 from Ph.D. Ko.
2.Transformation into E.coli BW25113
As the E.coli BW25113 is a host strain for the surface display and expression, we transformed the designed plasmid vector into it. We cultured the bacteria on M9 minimal media; one with glucose and another with cellobiose respectively. We checked the activity of cheater on M9 minimal medium with glucose and cooperator on M9 minimal medium with cellobiose.
3.Co-culture of cooperator and cheater with different ratio
Both cooperator and cheater were incubated in M9 minimal medium with 0.48% glucose at 37’C until it reached 1.0 in OD 600.
Batch co-culture was conducted in 5ml of M9 minimal medium with 0.48% cellobiose solution; in ratio of cheater and cooperator 1:1, 1:2, 1:4, 2:1, 4:1.
After 2, 4 and 6 days, each co-culture medium with different ratio was extracted and streaked on LB agar plate with ampicillin.
After overnight growth, total cell was counted and the plate was checked under fluorescence microscopy to count cheater; GFP expressing bacteria.