Team:NUS Singapore/Improvement
Part Improvement
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.
Characterisation Protocol:
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.
