Our team has decided to improve the BBa_K116404 phosphate sensor-GFP reporter which was constructed by NYMU Taipei in 2008. The part functions as an external phosphate ion sensor. When high phosphate concentration is present; the phosphate promoter pPhoB would be repressed and stop downstream GFP production.

By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed BBa_K2447000 phosphate sensor-GFP reporter. Our part is much more sensitive to phosphate concentrations even at phosphate concentrations of 40uM and above.

Transformed E. coli MG 1655 cells were incubated in LB broth with kanamycin (50 ng/µL) at 37 degree for 24 hours before being diluted 100x and then incubated for another 2-3 hours to reach an OD of 0.1. Cells were washed in MOPS medium (0.2% glucose) and subsequently re-suspended in MOPS (0.2% glucose). Next, cells are loaded into 96 well plate preloaded with various concentrations of phosphate concentrations. 10 mins interval reading of OD 600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at 37 degrees.

Figure 1: BBa_K2447000 Phosphate sensor coupled to GFP reporter. Strong GFP expression was elucidated for phosphate concentrations above 50uM.