1. Introduction

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.

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

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.

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.

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

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.

4. Materials and methods

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

3OC12HSL-dependent CmR expression

3OC12HSL-dependent C4HSL production

4-2. Assay Protocol

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

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

5. Reference