Construction of the 3OC12HSL-dependent 3OC6HSL production module

Fig2-1-3-1-5, Lux sender strain and Lux reporter strain

For construction of the 3OC12HSL-dependent 3OC6HSL production module, we firstly constructed a new part Plas-LuxI (BBa_K934012 ). Plas-LuxI cell is an engineered E.coli that contains a 3OC12HSL-dependent LuxI generator and a constitutive LasR generator. As the 3OC12HSL-dependent LuxI generator, we constructed a new Biobrick part Plas-LuxI (BBa_K934012 )by combining Plas promoter (BBa_K649000 ). and LuxI (BBa_K081008 ). As a constitutive LasR generator, we used Ptrc-LasR. By introducing Plas-LuxI and Ptrc-LasR into E.coli strain JM 2.300, we constructed Plas-LuxI cell. Then we performed a reporter assay by using Lux reporter cell to characterize the function of Plas-LuxI. As the negative control of 3OC6HSL production, we prepared 3OC6HSL non-producer cell (ΔP-LuxI cell) that contains, in addition to Ptrc-LasR, promoterless-LuxI (BBa_K081008) instead of Plas-LuxI (Fig2-1-3-1-5).

The ΔP-LuxI cell does not produce 3OC6HSL even though 3OC12HSL exist. The supernatants of the cultures of these modules were used as the inducer in the reporter assay (Fig2-1-3-1-6). We prepared four conditions as follow.

E) Culture containing Plas-LuxI cell without 3OC12HSL induction

F) Culture containing Plas-LuxI cell with 3OC12HSL induction

G) Culture containing ⊿P-LuxI cell without 3OC12HSL induction

H) Culture containing ⊿P-LuxI cell with 3OC12HSL induction

Fig2-1-3-1-6, How to perform 3OC12HSL-dependent 3OC6HSL production assay

Using the supernatant of the four culture conditions, we performed the reporter assay. In the reporter assay, we used a Lux reporter strain that contains Ptet-LuxR and Plux-GFP (BBa_K395100). 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.

Fig2-1-3-1-7, 3OC12HSL-dependent 3OC6HSL production

Fig2-1-3-1-7 shows fluorescence intensities by the reporter cells dependent on different conditions. Only when the supernatant of condition F was used, the fluorescence intensity of the Lux reporter cell increased while the supernatants of other three conditions did not affect. Comparing the results of the condition E and F, it can be said that with the induction of 3OC12HSL to Plas-LuxI, the fluorescence intensity of the Lux reporter cell increased by 112-folds. This result indicates that Plas-LuxI cell produced 3OC6HSL in response to 3OC12HSL induction by the function of Plas-LuxI (BBa_K934012) From this experiment, we confirmed that a new part Plas-LuxI (BBa_K934012) synthesized enough concentration of 3OC6HSL to induce the Lux reporter cell. [Protocol]