contents

1. Introduction

2. Summary of the Experiment

2.1. AHL dependent CmR expression

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 designed a signal-dependent growth system by using signaling molecules and antibiotic resistance gene. In our Prisoner’s Dilemma game (Fig. 3-1-1-1.), Prisoner A coli needs C4HSL and Prisoner B coli needs 3OC12HSL to acquire chloramphenicol resistance (CmR).
   As for Prisoner A, Pcon_rhlR_TT_Plux_CmR (pSB6A1) contains C4HSL-dependent chloramphenicol resistance gene product (CmR) generator and a constitutive rhlR generator. As a constitutive 3OC12HSL production module, we used Plac_lasI (pSB3K3).
   As for Prisoner B, Pcon_lasR_TT_Plux_CmR (pSB6A1) contains 3OC12HSL-dependent chloramphenicol resistance gene product (CmR) generator and a constitutive lasR generator. As a constitutive C4HSL production module, we used Plac_rhlI (pSB3K3).
   For precise implementation of our payoff matrix, we constructed improved parts Pcon_lasR_TT_Plux_CmRssrA, and Pcon_rhlR_TT_Plux_CmRssrA. The growth of the prisoner colis were confirmed by measuring the optical density.
  

Fig. 3-1-1-1. Our replicated payoff matrix for growth inhibition effect on Prisoner coli

Fig. 3-1-1-2. 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 realized.

Fig. 3-1-2-1. Cells for the experiment to realize 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. 3-1-2-2.). 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 realized.

Fig. 3-1-2-2. Cells for the experiment to realize 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 realized as the following.

Fig. 3-1-3-1. 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 realized as the following.

Fig. 3-1-3-2. 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. 3-1-4-1.

　　　　　　

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

Fig. 3-1-4-2.

　　　　　　

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

Fig. 3-1-4-3.

　　　　　　

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

Fig. 3-1-4-4.

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 (50 microg/mL) and Kan (30 microg/mL) 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.
   ①)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 50 microM C4HSL (30 microL) + Chloramphenicol (75 microg/mL)
   ②)LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (30 microL) + Chloramphenicol (75 microg/mL)
6.Grow the samples of cells at 37°C for more than 8 hours.
7.Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

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 (50 microg/mL) and Kan (30 microg/mL) 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 (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + 1 microM 3OC12HSL (3 microL) + Chloramphenicol (75 microg/mL)
   b) LB (3 mL) + antibiotics (Amp 50 microg/mL + Kan 30 microg/mL) + DMSO (3 microL) + Chloramphenicol (75 microg/mL)
6. Grow the samples of cells at 37°C for more than 8 hours.
   Measure optical density every hour. (If the optical density is over 0.9, dilute the cell medium to 1/5.)

5. Reference

　　　　　　

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