Team:NUS Singapore/Improvement
Part Improvement
Our Improved Part: Extracellular phosphate sensor with GFP reporter ✔Fully Functional
Preliminary modelling results revealed that the strength of the phosphate promoter is one of the highly sensitive parameter in the design. As the original phosphate construct BBa_K116404 (constructed by NYMU Taipei in 2008) is weak, we decided to improve the part by increasing the expression level and range to facilitate our building of the kill switch.
Hence, our team has decided to improve upon the original phosphate sensor-GFP reporter, by replacing the weaker RBS BBa_B0032 of the original part with the stronger RBS BBa_B0034, to augment the GFP expression. We have successfully constructed our version of the phosphate-sensor with GFP reporter BBa_K2447000 and had successfully improved upon the original part in terms of yielding greater GFP expression. 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).
Characterisation Protocol:
Transformed with E. coli MG 1655 cells were incubated in LB broth with kanamycin (50 ng/µL) at 37 °C for 24 hours before being diluted 100x and then incubated for another 2-3 hours to reach OD600 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 °C.
Figure 1: PhoR and PhoB proteins work in tandem to control promoter pPhoB and, in consequence, downstream expression of GFP. The part functions as an external phosphate ion sensor. When high phosphate concentration is present; the phosphate promoter pPhoB would be repressed and stopping downstream GFP production.
Figure 2: By replacing the weaker RBS BBa_B0032 with the stronger RBS BBa_B0034, our improved phosphate sensor with GFP reporter (BBa_K2447000) was able to elucidate higher GFP expression and was much more sensitive to varying concentration of phosphate ions.
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.
