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We created a symbiosis of Company and Customer for reproducing the situation in real economy. We used signaling molecules and antibiotics resistance gene, and constructed signal-dependent signal production in our system. In our bank story, we used signaling molecules 3OC12HSL as goods. For construction of the 3OC12HSL-dependent chloramphenicol resistance (CmR) and C4HSL production module, we constructed a new part Plux-CmR-RhlI (BBa_K1529797). Plux-CmR-RhlI cell is an engineered E. coli that contains a 3OC12HSL-dependent RhlI generator and a constitutive LuxR generator. As a constitutive LuxR generator, we used Ptet-LuxR. In our bank story, this part reproduces Customer. (Fig. 3-4-1-1.)　We confirmed that 3OC12-depedndent growth by measuring optical density, and 3OC12-dependent C4HSL production by using reporter cell.

Fig. 3-4-1-1. Customer’s Genetic Circuit

In the presence of 3OC12HSL, Customer express CmR and RhlI. (C4HSL) (Fig. 3-4-1-2.) (Fig. 3-4-1-3.)

Fig. 3-4-1-2. 3OC12HSL-dependent CmR expression assay Flow Chart

Fig. 3-4-1-3. 3OC12HSL-dependent C4HSL production assay Flow Chart

The supernatant of the customer cell were used as the inducer in the reporter assay.

2. Summary of the experiments

2-1. 3OC12HSL-dependent CmR expression

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring optical density of the cell cultures containing chloramphenicol.

In this experiment we prepared two plasmids, A and B. (See Fig. 3-4-2-1.) Concurrently with 3OC12HSL induction, we added chloramphenicol into the medium containing Customer cell and measured optical density for about 8 h to estimate the concentration of the cell.

Fig. 3-4-2-1. Plasmids for the experiment of 3OC12HSL-dependent CmR expression

2-2. 3OC12HSL-dpendent 4CHSL production

We performed a reporter assay by using reporter cells to characterize the function of 3OC12HSL-dependent C4HSL production. Plux-CmR-RhlI cells contain constitutive luxR generator, and produce C4HSL (RhlI) in the presence of 3OC12HSL. C4HSL is expressed to the culture, so the supernatant of the sender cell contains C4HSL. Reporter cells are incubated in the supernatant of the culture of sender cells. In the presence of C4HSL reporter cells express GFP. We checkefd the fluorescence of reporter cells to confirm of the expression of C4HSL.The expression of the reporter cells were confirmed by Flow Cytometer.

Fig. 3-4-2-2. Plasmids for the experiment of 3OC12HSL-dependent C4HSL production

We prepared four conditions as follow. (PlacIq-CmR cell were used as the negative control.)

A-1) Culture containing Plux-CmR-RhlI cell with 3OC12HSL induction

A-2) Culture containing Plux-CmR-RhlI cell witout induction

B-1) Culture containing PlacIq-CmR cell with 3OC12HSL induction

B-2) Culture containing PlacIq-CmR cell without induction

Reporter:

C) Culture containing constitutive RhlR generator and Prhl(RL)-GFP cell

D) Culture containing constitutive RhlR generator and PlacUV5-GFP cell…Positive control

E) Culture containing constitutive RhlR generator and Promoter-less-GFP cell…Negative control

3. Results

3-1. 3OC12HSL-Depemdent CmR Expression Assay

We got results of two type of culture condition which is difference in concentration of chloramphenicol. (Without chloramphenicol and 100 microg / ml)

Fig.3-4-3-1. shows every cell can grow in the absence of chloramphenicol. Conversely, Fig.3-4-3-2. shows some cells cannot grow in presence of chloramphenicol.

Fig. 3-4-3-1. 3OC12HSL-Dependent Customer growth in no Cm

Fig.3-4-3-2. 3OC12HSL-dependent Customer growth in 100 microg / mL Cm

With induction of 3OC12HSL, the cell containing Plux-CmR-RhlI can grow in the presence of chloramphenicol. However, without induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, only with induction of 3OC12HSL, Plux-CmR-RhlI cell can express CmR and grow well.

3-2. 3OC12HSL-Dependent C4HSL Production Assay

As Fig. 3-4-3-3. shows, when the reporter cells were incubated in the supernatant of the sender A-1, the fluorescence intensity of the reporter C increased. Comparing the results of used supernatant A-1 and A-2, reporter cell in the supernatant of A-1 (the induced Customer cell’s culture) had 95-fold higher fluorescence intensity.

This result indicates that Company cell produced C4HSL in response to 3OC12HSL induction by the function of Plux-CmR-RhlI.

From this experiment, we confirmed that a new part Plux-CmR-RhlI synthesized C4HSL (RhlI) as expected.

Fig.3-4-3-3. Customer excretes C4HSL when C12HSL exists

4. Materials and methods

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

3OC12HSL-dependent CmR expression

A. Ptet-GFP-Ptet-RhlR (psB6A1), Prhl(RL)-CmR-lasI(pSB3K3)

Fig. 3-4-4-1.

B. Ptet-GFP-Ptet-RhlR (psB6A1) PlacIq-CmR (pSB3K3) (Positive control)

Fig. 3-4-4-2.

3OC12HSL-dependent C4HSL production

Sender:

A. Ptet-LuxR-Plac-RFP(pSB6A1) Plux-CmR-RhlI(pSB3K3)

Fig. 3-4-4-3.

B. Ptet-LuxR-Plac-RFP(pSB6A1) Plux-CmR(pSB3K3) (Negative control)

Fig. 3-4-4-4.

Reporter:

C. Ptet-RhlR(pSB6A1) Plux-GFP(pSB3K3)

Fig. 3-3-4-5.

D. Ptet-RhlR(pSB6A1) PlacUV5-GFP(pSB3K3) (Positive control)

Fig. 3-4-4-6.

E. Ptet-RhlR(pSB6A1) Promoter-less-GFP(pSB3K3) (Negative control)

Fig. 3-4-4-7.

4-2. Assay Protocol

4-2-1. 3OC12HSL-depemdent CmR expression

1. Grow cell in LB containing antibiotic over night at 37°C.

2. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5 (→fresh culture)

3. Add 30 microL of suspension in the following medium.

1) 3 mL of LB containing Amp and Kan + 30 microL C4HSL (final concentration is 500 microM)

2) 3 mL of LB containing Amp and Kan + 30 microL DMSO

3) 3 mL of LB containing Amp, Kan and Cm (final concentration is 50mg/mL) + 30 microL C4HSL (final concentration is 500 microM)

4) 3 mL of LB containing Amp, Kan and Cm (final concentration is 50mg/mL) + 30 microL DMSO

4. Grow the samples of sender cells at 37°C for more than 10 hours.Measure optical density every hour. (If optical density is over 1.0, dilute the cell medium.)

4-2-2. 3OC12HSL-dependent C4HSL production

1. Prepare the supernatant of the sender cell

2. Grow the colony of sender cell in LB containing antibiotic O/N at 37°C.

3. Make a 1:100 dilution in 3 mL of fresh LB containing antibiotic and grow the cells at 37°C until the observed OD590 reaches 0.5.

4. Add 30 microL of the culture containing the cells in the following medium.

1) Add 30 microL of 500 microM 3OC12HSL to 3 mL LB containing Amp and Kan

2) Add 30 microL DMSO to 3 mL LB containing Amp and Kan

5 .Grow the samples of sender cell at 37°C for 8 hours.

6. Centrifuge sample at 9000x g, 4°C for 1minute.Filter sterilize supernatant. (Pore size is 0.22 microm. ) Use this supernatant in reporter assay.

Reporter Assay

1. Grow the colony of Reporter cell (described upper) in LB containing antibiotic (Amp and Kan) over night at 37°C.

2. Make a 1:100 dilution in 3 mL of fresh LB+ antibiotic and grow the cells at 37°C until you reach an 0.5 in OD590 (fresh culture).

3. Add 30 microL of the culture containing reporter cells in the following medium.

1) 2.7 mL filtrate of A① +600 microL LB

2) 2.7 mL filtrate of A② +600 microL LB

3) 2.7 mL filtrate of B① +600 microL LB

4) 2.7 mL filtrate of B② +600 microL LB

5) 2.7 mL filtrate of C① +600 microL LB

6) 2.7 mL filtrate of C② +600 microL LB

7) 3 mL LB + 500 microM C4HSL 30 microM (final concentration is 5 microM)

8) 3 mL LB + DMSO 30 microL

4. Grow the samples of Reporter cell in incubator at 37°C for 4 hours.

5. Start preparing the flow cytometer 1 h before the end of incubation.

6. After incubation, take the sample, and centrifuge at 9000x g, 1 min, 4°C.

7. Remove the supernatant by using P1000 pipette.

8. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend. (The ideal of OD is 0.3.)

9. Dispense all of each suspension into a disposable tube through a cell strainer.

10. Use flow cytometer to measure the fluorescence of GFP. (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company.)

5. Reference

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

Team:Tokyo Tech/Experiment/3OC12HSL-dependent C4HSL production