Team:Tokyo Tech/Experiment/Symbiosis confirmation by co-culture




Mutualism Confirmation ~Co-culture Assay~



1. Introduction

2. Summary of the Experiment

3. Results

4. Discussion

5. Materials and Methods

5-1. Construction

5-2 Assay Protocol

6. Reference



Mutualism Confirmation ~Co-culture Assay~


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.

Fig. 3-5-1-1. The genetic circuit of Company and Customer

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 six hours in the same medium, we detected the mutualism by checking the fluorescence intensity of GFP with flow cytometer. We measured the optical density every hour during the culturing.

Fig. 3-5-2-1. The flow chart of co-culture assay

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 = the optical density after co-culturing for 6 h x the ratio of the cells with the fluorescence of 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. The preliminary experiment results

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 that the proper condition in our experiment is adding 300 microL of Company cell and 100 microL of Customer cell to 2.6 mL of LB medium.(See Materials and methods 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 and Customer constructed a mutualistic relationship.


5. Materials and methods

5-1 Construction


All the samples were JM2.300 strain


A. Ptet-GFP-Ptet-RhlR (pSB6A1), Prhl(RL)-CmR-LasI (pSB3K3) ...Company

Fig. 3-5-5-1.

B. Ptet-LuxR-Plac-RFP (pSB6A1), Plux-CmR-RhlI (pSB3K3) ...Customer

Fig. 3-5-5-2.


5-2 Assay Protocol

1. Prepare overnight cultures for each samples A and B in 3 mL LB medium, containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) at 37°C for 12 h.
2. Dilute the overnight cultures to 1 / 100 in fresh LB medium (3 mL) containing ampicillin (50 microg / mL) and kanamycin (30 microg / mL) [fresh culture].
3. Incubate the fresh cultures in 37°C until the observed OD590 reaches 0.5.If the OD becomes over 0.5, dilute to 0.5 with LB medium.
4. Add the culture to LB medium as below.
  LB medium contains 50 microg / mL ampicillin, 30 microg / mL kanamycin and 100 microg / mL chloramphenicol.
  • A 300 microL + B 130 microL+ LB medium 2.57 mL
  • A 300 microL + LB medium 2.7 mL
  • B 130 microL + LB medium 2.87 mL
5. Incubate these samples at 37°C for 6 h. (During that time, measure the optical density every one hour.)
6. Measure the fluorescence intensity with a flow cytometer (We used BD FACSCaliburTM Flow Cytometer of Becton, Dickenson and Company).

6. Reference

1. Bo Hu et al. (2010) An Environment-Sensitive Synthetic Microbial Ecosystem. PLoS ONE 5(5): e10619