1. Introduction

We created a symbiosis of Company E. coli and Customer E. coli 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 C4HSL as money. For construction of the C4HSL-dependent chloramphenicol resistance (CmR) and 3OC12HSL production module, we designed 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 is Company. (Fig. 3-3-1-1.)

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

To confirm function of Company cell, we performed two kinds of assays. One is C4HSL-Dependent CmR Expression Assay. In this experiment we prepared four plasmid sets (A, B, C, D) shown in below. (Fig. 3-3-2-1.) Concurrently with C4HSL induction, we added chloramphenicol into the medium containing Company cell and measured optical density for about 8 h to estimate the concentration of the cell. The other is C4HSL-Dependent 3OC12HSL Production Assay. In this experiment we prepared three more plasmid sets (E, F, G) shown in below. (Fig. 3-3-2-1.) First, we added C4HSL to the culture of sender cell, and induced the expression of LasI. Then, the supernatants of the culture were used as the inducer in the reporter assay. We measured the expression of GFP in reporter cells by flow cytometer.

We prepared four conditions as follow.

3. Result

3-1. C4HSL-Dependent CmR Expression Assay

After induction, optical densities were measured to estimate the concentration of the cell. We prepared two types of culture conditions which is different in concentration of chloramphenicol. (Without chloramphenicol and 100 microg/ml)

Fig.3-3-3-1. shows every cell can grow in the absence of chloramphenicol. Conversely, Fig.3-3-3-2. shows some cells cannot grow in presence of chloramphenicol. With induction of C4HSL, the cell containing Prhl(RL)-CmR-LuxI can grow in the presence of chloramphenicol. (The growth curve of the cell is same as the growth curve of positive control.) However, without induction of C4HSL, the cell cannot express CmR and cannot grow in the presence of chloramphenicol. (The growth curve of the cell is same as the growth curve of negative control.) As a result, only with induction of C4HSL, Prhl(RL)-CmR-LuxI (BBa_K1529302) cell can express CmR and grow well.

3-2. C4HSL-Dependent 3OC12HSL Production Assay

Four hours after addition of the supernatants from the culture of sender cells, we measured the expression of GFP in the reporter cells by flow cytometer.

As Fig. 3-3-3-3. shows, when the supernatant of condition ??? was used, the fluorescence intensity of the reporter cell increased. Comparing the results of condition ??? and ???, reporter cell in the supernatant of induced Company cell culture had the ???-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 these experiments, we confirmed that a new part Prhl(RL)-CmR-LasI (BBa_K1529302) synthesized CmR and 3OC12HSL in the presence of C4HSL.

4. Materials and methods

4-1 Construction

-Strain

All the samples were JM2.300 strain.

-Plasmids

4-2. Assay Protocol

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

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

5. Reference