Team:Tokyo Tech/Parts

Best new Basic part: BBa_K1529300, BBa_K1529310, BBa_K1529320

• Prhl(RL) : BBa_K1529300
• Prhl(LR) : BBa_K1529310
• Prhl(RR) : BBa_K1529320

Prhl(RL) Promoter has the biggest ratio of leakage and expression (when C4HSL is induced) among the three above.

Best new Composite part: BBa_K1529301, BBa_K1529302, BBa_K1529797

Prhl(RL)_GFP (BBa_K1529301)

Prhl(RL) is a promoter that is activated by C4HSL. 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).

Prhl(RL)_CmR_LasI (BBa_K1529302)

We constructed this part by combining BBa_K1529300, BBa_K395160, BBa_B0034 and BBa_C0078.
(CmR is Chloramphenicol resistance.)

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. This is the first Biobrick part that succeeded in C4HSL dependent CmR and 3OC12HSL　production. Using this part with the plasmid that is constitutively expressing RhlR, we succeeded in confirming RhlR activates the expression of CmR and LasI in the presence of C4HSL.

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.

Best Part Collection: BBa_K1529302, BBa_K1529797, BBa_K1529263

Plux_CmR_LasI (BBa_K1529263)

We constructed this part by combining BBa_K395162, BBa_B0034 and BBa_C0078.
(CmR is Chloramphenicol resistance.)

To characterize Plux-CmR-LasI (BBa_K1529263), we introduced Plux-CmR-LasI on pSB3K3 with Ptet-RhlR-Ptet-GFP on pSB6A1 to E.coli as “C4HSL dependent CmR and 3OC12HSL producer cell”. In this E.coli, constitutively expressed RhlR activates the expression of CmR and LasI in the presence of C4HSL.