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.
Figure 1: PhoR and PhoB proteins work in tandem to control promoter pPhoB and, in consequence, downstream expression of GFP
By replacing the weaker RBS BBa_B0032 of the original part with a stronger RBS BBa_B0034, we have successfully constructed BBa_K2447000 an improved phosphate sensor-GFP reporter. Our part shows, on average, 40 fold increase in GFP expression (Figure 3) when compared to the previous version of the construct. The original phosphate construct is also insensitive to high phosphate concentrations above 50 µM where similar levels of GFP expression are observed (Figure 4). Unlike the previous construct, our improved phosphate construct is much more sensitive to various phosphate concentrations from 0 to 1000 µM, particularly at phosphate concentrations above 50 µM (Figure 3).
Figure 2: By replacing the weaker RBS BBa_B0032 with the stronger RBS BBa_B0034, our improved phosphate sensor with GFP reporter (BBa_K2447000) is able to elucidate higher GFP expression and is much sensitive to varying concentration of phosphate ions.
Characterisation Protocol:
Transformed with ? E. coli MG 1655 cells were incubated in LB broth with kanamycin (50 ng/µL) at 37 degrees for 24 hours before being diluted 100x and then incubated for another 2-3 hours to reachOD600 of 0.1. Cells were washed in MOPS medium (0.2% glucose) and subsequently re-suspended in MOPS (0.2% glucose). Next, cells were loaded into 96 well plate preloaded with various concentrations of phosphate concentrations. 10 mins interval reading of OD600 and GFP absorbance was conducted over a continuous 8 hours run of the microplate reader at 37 degrees.
Figure 3: Side by side comparison of the improved phosphate construct (BBa_K2447000) and original phosphate construct (BBa_K116404) in terms of GFP expressions.
Figure 4: Original phosphate construct (BBa_K116404) designed by the Taiwanese iGEM team in 2008.
