Designed by: Ma Tsz Shan Group: iGEM13_Hong_Kong_HKU (2013-09-15)
pT7-ppk1(K.oralis)
Polyphosphate kinase (ppk1) (E.C. 2.7.4.1) catalyzes the formation of polyphosphate from ATP. In BBa_K1217003, ppk1 was under the control of T7 promotor. Under iptg induction, over-expression of ppk1 can reduce the phosphate level in the medium by its incorporation into polyphosphate synthesis inside the bacteria.
Usage and Biology
Efficiency of this construct is compared with Two other designs BBa_K1217008 and BBa_K1217010.
We test the constructs' efficiency from 2 parameters: poly-P synthesis efficiency and phosphate removal efficiency from the medium.
Expected model of BBa_K1217003
Expected model of BBa_K1217008
Expected model of BBa_K1217010
Expression of BBa_K1217003
We confirm the expression of PPK1 enzyme in e.coli.
SDS-PAGE analysis of expression of K.oralis PPK1 enzyme inside E.coli. Expression of PPK1 is induced by 0.5mM IPTG at 30 degree for 5 hours. Band marked with star is the PPK1 enzyme, the enzyme expressed is soluble.
Efficiency of poly-P synthesis and phosphate removal from environment
Experiment procedure. Wide type and blank act as a control to indicate the normal phosphate uptake of wide type e.coli and the phosphate level change along with time. Upon iptg induction, cultural samples are collected in every three hours. The cell pellet harvested is used to measure the intracellular poly-P level and the supernatant is used for phosphate quantification
Intracellular poly-P quantification
Intracellular poly-P is extracted from the cells and quantified using DAPI fluorescence measurement. DAPI is a fluorescent probe that binds DNA and poly-P. Since DNA in the sample has been removed during poly-p extraction step, it reduce nonspecific fluorescent signal due to DNA-DAPI binding. Poly-P standard curve is shown.
Intracellular Poly-P level of all samples. Blue: Wild type indicate the basal poly-P level inside wild type cell. Red: BBa_K1217003. Black: BBa_K1217010. Green:BBa_K1217008. All 3 constructs have higher poly-P amount accumulated.
Phosphate level in the medium
Supernatant medium collected at each time point is quantified using Malachite green colorimetric assay. Phosphate standard curve is shown.
Phosphate level in medium. Blue: Wild type indicate the basal phosphate removal from the medium by wild type cell. Red: BBa_K1217003. Green: BBa_K1217008. The two constructs have higher phosphate removal from the medium.
