Team:Tokyo Tech/Experiment/C6


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Construction of the 3OC6HSL-dependent 3OC12HSL production module

Fig2-1-3-1-2, Las sender strain and Las reporter strain

For construction of the 3OC6HSL-dependent 3OC12HSL production module, we firstly constructed a new part Plux-LasI (BBa_K934022). Plux-LasI cell is an engineered E.coli that contains a 3OC6HSL-dependent LasI generator and a constitutive LuxR generator. As a 3OC6HSL-dependent LasI generator, we constructed a new Biobrick part Plux-LasI (BBa_K934022) by combining Plux promoter (BBa_R0062) and LasI (BBa_K081016 ). As a constitutive LuxR generator, we used Ptet-LuxR (BBa_S03119). By introducing Plux-LasI (BBa_K934022 ) and Ptet-LuxR (BBa_S03119) into E.coli strain JM2.300, we constructed Plux-LasI cell. Then we performed a reporter assay by using Las reporter cell to characterize the function of Plux-LasI (BBa_K934022) . As the negative control of 3OC12HSL production, we prepared 3OC12HSL non-producer cell (ΔP-LasI cell) that contains, in addition to Ptet-LuxR (BBa_S03119), promoterless-LasI (BBa_K081016) instead of Plux-LasI (BBa_K934022) (Fig2-1-3-1-2).

The ⊿P-LasI cell does not produce 3OC12HSL even though 3OC6HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-3). We prepared four conditions as follow.

A) Culture containing Plux-LasI cell without 3OC6HSL induction

B) Culture containing Plux-LasI cell with 3OC6HSL induction

C) Culture containing ⊿P-LasI cell without 3OC6HSL induction

D) Culture containing ⊿P-LasI cell with 3OC6HSL induction

Fig2-1-3-1-3, How to perform 3OC6HSL-dependent 3OC12HSL production assay

Using the supernatant of the four culture conditions, we performed the reporter assay.

In the reporter assay, we used a Las reporter strain that contains Ptrc-LasR and Plas-GFP (BBa_K649001). Also, a reporter cell that expresses GFP constitutively and a reporter cell that does not express GFP were used as the positive control and the negative control, respectively.

Fig2-1-3-1-4, 3OC6HSL-dependent 3OC12HSL production

Fig2-1-3-1-4 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition B was used, the fluorescence intensity of the Las reporter cell increased, while the supernatants of other three conditions did not affect. Comparing the results of the condition A and B, it can be said that with the induction of 3OC6HSL to Plux-LasI cell, the fluorescence intensity of the LasI reporter cell increased by 20-folds. This result indicates that Plux-LasI cell produced 3OC12HSL in response to 3OC6HSL induction by the function of Plux-LasI (BBa_K934022 ). From this experiment, we confirmed that a new part Plux-LasI (BBa_K934022 ) synthesized enough concentration of 3OC12HSL to induce the Las reporter cell. [Protocol]