1. Introduction

We designed signal-dependent signal production in our system by using signaling molecules and antibioticss resistance gene. In our bank story, we used signaling molecules C4HSL as money.

For construction of the C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we constructed a new part Prhl(RL)-CmR-LasI(BBa_K1529302). Prhl(RL)-CmR-LasI cell is an engineered E. coli that contains a C4HSL-dependent LasI generator and a constitutive RhlR generator. As a constitutive RhlR generator, we used Ptet-RhlR. In our bank story, this part imitates the functions of Company. (Fig. 3-3-1-1.) We confirmed that C4HSL-depedent growth by measuring optical density, and C4HSL-dependent 3OC12HSL production by using reporter cell.

In order to confirm Company’s dependency on C4HSL, we measured the growth of Company cell and expression of 3OC12HSL in the presence and absence of C4HSL.

2. Summary of the experiments

2-1. C4HSL-dependent CmR expression

We confirmed the function of C4HSL-dependent CmR expression by measuring optical density of the cell cultures containing chloramphenicol.

In this experiment we prepared three plasmids, A, B and C. (See Fig. 3-4-2-1.) Right after the C4HSL induction, we added chloramphenicol into the medium containing Company cell. We measured optical density for about 8 h to estimate the concentration of the cell.

2-2. C4HSL-dependent 3OC12HSL production

We performed a reporter assay by using reporter cells to characterize the function of C4HSL-dependent C4HSL production. Prhl(RL)-CmR-LasI cell containing constitutive RhlR generator expresses LasI and produces 3OC12HSL in the presence of C4HSL. Since 3OC12HSL is excreted to the culture, the supernatant of the sender cell contains 3OC12HSL when the part works as expected.

Reporter cells are incubated in the supernatant of the culture of sender cells. When there are 3OC12HSL in the supernatant, reporter cell expresses GFP. We checked the fluorescence of reporter cells to confirm of the expression of 3OC12HSL.The expression of the reporter cells were measured by Flow Cytometer.

We prepared the following conditions for the induction of reporter cells. (Plux-CmR cell was used as the negative control.)

(1) Culture containing Prhl(RL)-CmR-lasI cell with C4HSL induction

(2) Culture containing Prhl(RL)-CmR-lasI cell without induction

(3) Culture containing Plux-CmR cell with C4HSL induction

(4) Culture containing Plux-CmR cell without induction

(5) 5 microM of artificial C4HSL in LB medium

(6) DMSO in LB medium

Reporter

3. Result

3-1. C4HSL-dependent CmR expression assay

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

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

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

On the other hand, in the presence of chloramphenicol, the cell containing Prhl(RL)-CmR-LasI can grow only when induced by C4HSL. Without the induction of C4HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. As a result, we confirmed that Prhl(RL)-CmR-LasI expressed CmR when induced by C4HSL as expected.

3-2. C4HSL-dependent 3OC12HSL production Assay

Fig. 3-3-3-3. shows the fluorescence intensities generated by reporter cells. When the reporter cell E was incubated in the condition (1) (the culture of the induced Company 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 29-fold higher fluorescence intensity.

This result indicates that Company cell produced 3OC12HSL in response to C4HSL induction by the function of Prhl(RL)-CmR-LasI.

From this experiment, we confirmed that a new part Prhl(RL)-CmR-LasI synthesized 3OC12HSL (LasI) as expected.

4. Materials and methods

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

--C4HSL-dependent CmR expression

4-2. Assay Protocol

4-2-1. C4HSL-Dependent CmR Expression Assay

4-2-2. C4HSL-Dependent 3OC12HSL Production Assay

5. Reference