Team:Manchester/Description
Project Description
Polyphosphate Kinase (PPK) Fusion Protein
Basic aim: Assess impact localisation tag has on kinetics of PPKs
Introduction
Our project initially aimed to screen a selection of bacterial polyphosphate kinases to select one with the greatest forward rate of reaction (polyphosphate formation). Due to time constraints, we have decided to focus on a single polyphosphate kinase (PPK) that we identified in the Brenda database. We chose a PPK from Corynebacterium glutamicum, a bacteria used for industrial production of amino acids. The bacteria was discovered to accumulate high concentrations of polyphosphate (600mM, 37% cell volume), which was largely attributed to its class II Polyphosphate kinase. This PPK (encoded by NCgl2620) has a Kcat value of 74 for ATP ➝ Poly P under the experimental conditions used. It is also fully soluble, unlike the E. coli PPK which was encapsulated within a Pdu microcompartment by Warren et al. earlier this year. We hypothesise that solubility may enhance efficiency of encapsulation.
At this stage we will only be trialing one localisation tag (PduD1-20) and hope to characterise it using mCherry and fluorescence microscopy as an indicator for incorporation into microcompartment.
