1. Introduction

We designed a signal-dependent signal production in our system by using signaling molecules and antibiotics resistance gene. In our bank story, we used signaling molecule 3OC12HSL as product.

For construction of the 3OC12HSL-dependent chloramphenicol resistance gene product(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 3OC12HSL-dependent RhlI generator and a constitutive LuxR generator. As a constitutive LuxR generator, we used Ptet-LuxR. In our bank story, this part imitates the function of Customer. (Fig. 3-4-1-1)　We confirmed the 3OC12HSL-dependent growth by measuring the optical density, and 3OC12HSL-dependent C4HSL production by using reporter cell.

2. Summary of the experiments

2-1. 3OC12HSL-dependent CmR expression

We confirmed the function of 3OC12HSL-dependent CmR expression by measuring the optical density of the cell cultures containing chloramphenicol.(Fig. 3-4-2-1)

In this experiment, we prepared two plasmids A and B (See Fig. 3-4-2-2).Right after the 3OC12HSL induction, we added chloramphenicol into the medium including Customer cell. We measured the optical density for about eight hours to estimate the concentration of the cell.

2-2. 3OC12HSL-dependent C4HSL production

We performed a reporter assay by using reporter cell C, D and E to characterize the function of 3OC12HSL-dependent C4HSL production. Plux-CmR-RhlI cell containing constitutive LuxR generator expresses RhlI and produces C4HSL (RhlI) in the presence of 3OC12HSL. Since C4HSL is excreted to the culture, the supernatant of the sender cell contains C4HSL when this part works as expected.

The reporter cell was incubated in the supernatant of the culture of the sender cell. When there are C4HSL in the supernatant, the reporter cell expresses GFP.(Fig. 3-4-2-3) We checked the fluorescence intensity of the reporter cell to confirm the production of C4HSL. The fluorescence intensity of the reporter cell was measured by flow cytometer.

We prepared the following conditions for the induction of the reporter cells. (PlacIq-CmR cell was used as the negative control of RhlI. See Fig.3-4-2-4)

Reporter cells

3. Results

3-1. 3OC12HSL-dependent CmR expression assay

We tested two types of culture condition which contains different concentration of chloramphenicol(Cm). (0 and 100 microg / mL)

Fig. 3-4-3-1 and Fig. 3-4-3-2 show the condition in the absence and the presence of chloramphenicol, respectively.

Fig. 3-4-3-1 shows that every cell can grow in the absence of chloramphenicol.

On the other hand, in the presence of chloramphenicol, the cell containing Plux-CmR-RhlI can grow only when it was induced by 3OC12HSL. Without the induction of 3OC12HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Plux-CmR-RhlI expressed CmR when induced by 3OC12HSL as expected.

3-2. 3OC12HSL-dependent C4HSL production assay

Fig. 3-4-3-3 shows the fluorescence intensities generated by reporter cells. When the reporter cell C (Plux-CmR-RhlI) was incubated in the condition (1) (the culture of the induced Customer cell), the fluorescence intensity of the reporter cell increased. Comparing the results of condition (1) and (2), reporter cell in the supernatant of (1) had 95-fold higher fluorescence intensity.

This result indicates that Customer 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