1.Introduction

From the results so far, Company cell containing Prhl(RL)-CmR-LasI(BBa_K1529302) expresses CmR and LasI in the presence of C4HSL. This enables Company to survive and to produce 3OC12HSL. (This result is showed here.) On the other hand, Customer cell containing Plux-CmR-RhlI(BBa_K1529797) expresses CmR and RhlI in the presence of 3OC12HSL. This enables Customer to survive and to produce C4HSL. (This result is showed here.) These functions will make the mutualism of Company and Customer possible.

2.Summary of the experiment

To characterize the mutualism of Company cell and Customer cell, we cultivated both of them in different ratio and measured the growth. We constructed the Company cell containing GFP and the Customer cell containing RFP. After cultivating both of them for 6 hours in the same medium, we detected the mutualism and its condition by checking the fluorescence intensity of GFP with flow cytometer. We measured optical density every hour during the culturing.

Fig. 3-5-2-1. The flow chart of co-culture assay図があってないけどどうしましょう…updateする？

3. Results

Fig. 3-5-3-1. The growth of the two cells when co-cultured

Fig.3-5-3-1. shows the optical densities of Company and Customer after they were co-cultured for 6 hours. These values were evaluated below:

The optical density of Company cell = after co-culture for 6 h x the ratio of the cells fluorescing by GFP.

The optical density of Customer cell = the optical density after co-culturing for 6 h x the ratio of the cells without fluorescence of GFP.

As a result, the co-cultured samples were able to grow better than the samples single cultured . It means that only Customer cell can grow little by little in the medium with 100 microg/mL Chloramphenicol because of the leak of Plux promoter (Company cell can’t grow since it contains Prhl promoter which has less leak.) However, in the co-culture experiment, Customer produced C4HSL by the leakage and it induced CmR and LasI expression of Company. LasI produced 3OC12HSL, and it induced CmR and RhlI expression of Customer. This positive feedback might cause the mutualism of Company and Customer.

4. Discussion

Before conducting this experiment, we checked whether the mutualism could be achieved by mathematical modeling (Go to modeling page.) This modeling results showed that the mutualism between the Company and Customer is significantly affected by the initial cell concentration. Therefore, we conducted this assay with the following conditions as preliminary experiment. The experimental results are shown below.

Fig. 3-5-4-1 shows that the mutualism cannot be achieved when the initial population density is too low. Also, when the initial population density is too high, the mutualism cannot be characterized because of reaching a saturation of population before the measurement.

Additionally, it was found that Customer could survive in the presence of chloramphenicol even when its initial population density was low(Go to the 3OC12HSL-dependent growth experiment page).

Based on these results, we estimated the proper condition of 300 microL of Company cell and 100 microL of Customer in our experiment(See experiment page for the detailed procedures).

So we characterized the mutualism between Company and Customer with the following conditions.

Fig. 3-5-4-2. Detailed result of Co-culture assay

Fig. 3-5-4-3. Optical Density

Fig. 3-5-4-2 revealed that when the amount of Company is 300 microL, the mutualism can be achieved in the range of 60 to 200 microL of Customer.

Fig. 3-5-4-3 shows the time-dependent change of optical density. The growth of the cells in mutualism was faster than that of in single culture.

From these results described above, our Company E. coli and Customer E. coli constructed a mutualistic relationship.

5. Materials and methods

5-1 Construction

-Strain

All the samples were JM2.300 strain

-Plasmids

Fig. 3-5-5-1.

Fig. 3-5-5-2.

5-2 Assay Protocol

6. Reference