contents

1. Introduction

2. Summary of the Experiment

2.1. Replicating the Payoff Matrix of Prisoner A coli

2.2. Replicating the Payoff Matrix of Prisoner B coli

3. Results

3.1. Replicating the Payoff Matrix of Prisoner A coli

3.2. Replicating the Payoff Matrix of Prisoner B coli

4. Materials and Methods

4.1. Construction

4.2. Assay Protocol

4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

4.2.2. 3OC12HSL concentration-dependent CmR expression assay

5. Reference

1. Introduction

　　　　　　

We genetically engineered two prisoner coli, Prisoner A and Prisoner B. They are able to cooperate or to defect. The genetic circuits, with the improved chloramphenicol resistant protein(CmR) part, of Prisoner A and B are shown in Fig. 3-2-1-1. Our goal in this project is to replicate the payoff matrix (Fig. 3-2-1-2) with the four types of growth inhibition. Using the improved plasmids we constructed, our E.coli version payoff matrix is replicated through wet experiments.

Fig. 3-2-1-1. The genetic circuits of prisoner colis with the options of cooperation or defection

Fig. 3-2-1-2. Payoff Matrix of Prisoner A and B

　　　

2. Summary of the Experiment

2.1. Replicating the Payoff Matrix of Prisoner A coli

The cell growth of the Prisoner colis containing the pairs of plasmids (1) and (2) in the lower chloramphenicol (Cm) concentration (75 microg/mL) were measured and the payoff matrix was replicated.

Fig. 3-2-2-1. Cells for the experiment to replicate the payoff matrix of Prisoner A coli

2.2. Replicating the Payoff Matrix of Prisoner B coli

We have prepared a new pair of plasmids, (3) and (4), as in the following (Fig. 3-2-2-2.). The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and also in the lower 3OC12HSL concentration were measured and the payoff matrix was replicated.

Fig. 3-2-2-2. Cells for the experiment to replicate the payoff matrix of Prisoner B coli

　　　　　　

Using the pair of plasmids we constructed, our E.coli version payoff matrix with the four types of growth inhibition was replicated through wet experiments. The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4), grown under the lower Cm concentration and also with the lower 3OC12HSL concentration, were observed.

3. Results

3.1. Replicating the Payoff Matrix of Prisoner A coli

The cell growth of the Prisoner colis containing the pairs of plasmids (1) and (2) in the lower chloramphenicol (Cm) concentration (75 microg/mL) were measured after an eight hour incubation. The payoff matrix was replicated as the following.

Fig. 3-2-3-1. Payoff matrix of Prisoner A coli with 75 microg/mL Cm

3.2. Replicating the Payoff Matrix of Prisoner B coli

　　　　　　

The cell growth of the Prisoner colis containing the pairs of plasmids (3) and (4) in the lower chloramphenicol (Cm) concentration (75 microg/mL) and in the lower 3OC12HSL concentration were measured. The payoff matrix was replicated as the following.

Fig. 3-2-3-2. Payoff matrix of Prisoner B coli with gfp in 75 microg/mL Cm and 1 nM 3OC12HSL

4. Materials and Methods

4.1. Construction

-Strain

　　　　　　

All the samples were JM2.300 strain.

-Plasmids

　　　　　　

(1) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + Plac_lasI (pSB3K3)

Fig. 3-2-4-1.

　　　　　　

(2) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) + promoter less_lasI (pSB3K3)

Fig. 3-2-4-2.

　　　　　　

(3) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + Pcon_gfp_rhlI (pSB3K3)

Fig. 3-2-4-3.

　　　　　　

(4) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) + promoter less_rhlI (pSB3K3)

Fig. 3-2-4-4.

4.2. Assay Protocol

4.2.1. C4HSL-dependent CmR expression assay ([Cm] = 75 microg/mL)

-samples
(1) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) ＋ Plac_lasI (pSB3K3)
(2) Pcon_rhlR_TT_Plux_CmRssrA (pSB6A1) ＋ promoter less_lasI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB (containing Amp and Kan) and grow the cells at 37°C until the observed OD₅₉₀ reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB (containing Amp and Kan).
5. Add 30 microL of suspension in the following medium.
   a)LB (containing Amp and Kan) (3 mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (75 microg/mL)
   b)LB (containing Amp and Kan) (3 mL) + DMSO (30 microL) + Chloramphenicol (75 microg/mL)
6.Incubate the samples of cells at 37°C for more than 8 hours.
7.Measure OD₅₉₀ every hour.

4.2.2. 3OC12HSL concentration-dependent CmR expression assay

-samples
(3) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) ＋ Pcon_gfp_rhlI (pSB3K3)
(4) Pcon_lasR_TT_Plux_CmRssrA (pSB6A1) ＋ promoter less_rhlI (pSB3K3)

-Procedure
1. Prepare overnight cultures (n=2) for the samples in 3 mL LB medium, containing ampicillin (50 microg/mL) and kanamycin (30 microg/mL) at 37°C for 12 hours.
2. Make a 1:100 dilution in 3 mL of fresh LB containing Amp and Kan and grow the cells at 37°C until the observed OD₅₉₀ reaches 0.5.
3. Centrifuge 1 mL of the sample at 5000g, RT for 1 minute.
4. Suspend the pellet in 1mL of LB containing Amp and Kan.
5.Add 30 microL of suspension in the following medium.
   a) LB (containing Amp and Kan) (3 mL) + 5 microM 3OC12HSL (0.6 microL) + Chloramphenicol (75 microg/mL)
   b) LB (containing Amp and Kan) (3 mL) + DMSO (0.6 microL) + Chloramphenicol (75 microg/mL)
6. Incubate the samples of cells at 37°C for more than 8 hours.
   Measure OD₅₉₀ every hour.

5. Reference

　　　　　　

1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619