We studied the game theory in order to plan further experiments on it.
Additionally, we looked through previous igem team projects regarding the game theory.
We discussed how to conduct the experiments.
In addition, we collected several protocols that are required for experiments.
We received cell stocks from Hyeok-jin Ko Ph.D for our experiments.
We began preparation for experiments. Ex. ampicillin containing plates.
We discussed with our instructor and professor to design our submitting parts.
We carried out mini-prep on beta-glucosidases for cooperator and selected sde_3603 among them.
After gel electrophoresis, we found out that mini-prep sde_3603 was contaminated, so we did the mini-prep once again but with some modifications.
We transformed the second mini-prep sde_3603 and streaked on ampicillin plates; one with 50ul and one with 10 fold diluted 100ul.
We made M9 minimal medium for later use; cooperator and cheater growth.
We streaked on plates from the given cell stocks to get colonies, but on the first trial we could not find any so we carried out once again.(used competent cell: E.coli BW25113)
Cheater and Cooperator streaking from cell stock 1: could not find colonies
Cheater from cell stock again
We did mini-prep for GFP and checked its existence by gel electrophoresis. Then, we transformed the plasmid into DH5α.
We streaked both cooperator and cheater on M9 minimal medium to check its growth.
We found colonies on cheater plate, but no colonies on cooperator plate. We streaked cooperator again on a new M9 minimal medium.
We did mini-prep from dh5a that we transformed GFP in previous week.
We checked the activity of GFP; cheater with UV light and it was successful.
We made M9 minimal medium with cellobiose rather than dextrose to check the activity of beta-glucosidase; cooperator.
We checked the sequence of both plasmids to create submitting parts.
We checked the activity of beta-glucosidase by culturing the cooperator on M9 minimal medium containing cellobiose and it was successful. We also checked the activity of GFP on the same medium to see if the cheater grows on cellobiose containing medium where it should not be grown.
We planned how to co-culture the cheater and cooperator; different concentration ratio and glucose addition.
We made M9 minimal medium with both 0.48% cellobiose and glucose respectively.
We inoculated both cheater and cooperator in M9 minimal medium with glucose to reach 1 at OD600. As they did not grow at all overnight, we made the medium once again.
It took us more than 24 hours for bacteria to grow. We inoculated once again the grown bacteria in 5ml into the 200ml M9 minimal medium with glucose.
We prepared for part submission; have done PCR with the insert and have done gel preparation for DNA purification. It was successful.