Team:Tokyo Tech/Parts

1. Best New Basic Part: BBa_K1529301, BBa_K1529311, BBa_K1529321

• Prhl(RL)-GFP (BBa_K1529301)

Prhl(RL) is a promoter that is activated by C4HSL-RhlR complex. 
We improved Prhl promoter (BBa_R0071) by changing LuxR binding site of Plux promoter (BBa_R0062) to RhlR binding site.
To characterize the function of the Prhl(RL) promoter (BBa_K1529300), we constructed this part,  Prhl(RL)-GFP (BBa_K1529301) , by inserting a Prhl(RL) promoter into the upstream region of GFP coding sequence .

By using reporter cells containing Prhl(RL)-GFP, we measured the fluorescence intensity of the cells induced by C4HSL. 
We confirmed that our new Prhl(RL) promoter was actually activated by C4HSL through an induction assay. (Fig. 5-1-1-1.)

2. Best New Composite Part: BBa_K1529302, BBa_K1529797

•Prhl(RL)-CmR-LasI (BBa_K1529302)

We constructed this part by combining  BBa_K1529300, BBa_K395160, BBa_B0034 and BBa_C0078. Prhl(RL) promoter has no leakage and higher expression than Prhl promoter (BBa_R0071). CmR and LasI are inserted into the downstream of the promoter

To confirm CmR production, we added chloramphenicol into the medium containing Prhl(RL)-CmR-LasI cell and measured optical density for about 8 h to estimate the concentration of the cell. (Fig. 5-1-2-1.)

Moreover, by co-culturing the cells containing this part with the cells containing 3OC12HSL dependent C4HSL producer part (BBa_K1529797), we succeeded in constructing a positive feedback system.

• Plux-CmR-RhlI (BBa_K1529797)

We constructed this part by combining BBa_K395162, BBa_B0034 and BBa_C0070. (CmR is Chloramphenicol resistance.)
This is the first BioBrick part that succeeded in 3OC12HSL dependent CmR and C4HSL　production. In our experiment, we used 3OC12HSL instead of 3OC6HSL as an inducer in order to circumvent crosstalk. Using this part with the plasmid that is constitutively expressing LuxR, we succeeded in confirming LuxR activates the expression of CmR and RhlI in the presence of 3OC12HSL.

To confirm C4HSL production, we measured the expression of GFP in reporter cells by flow cytometer. (Fig. 5-1-2-2).

3. Best Part Collection: BBa_K1529302, BBa_K1529797

Using these parts enable us to accomplish mutualism. The results of the co-culture assay is shown in Fig. 5-1-2-3. Company’s characteristics are C4HSL-dependent survival and 3OC12HSL production, and Customer’s characteristics are the opposite from Company’s. From these characteristics, the symbiosis between the two cells can be established. Two types of fluorescent proteins were used to trace the growth of each cells in our symbiosis experiments. We constructed the Company cell containing GFP and Customer cell containing RFP. By measuring the OD of the cells expressing GFP with flow cytometer, the symbiosis was detected. The result of the co-culture assay is shown in Fig. 2-1-16. By looking at the Company cells expressing GFP, the OD increased with the co-cultured cells than Company cells. From this point, we can say that Company and Customer actually mutualize in the medium.