Difference between revisions of "Team:Manchester/Description1"

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<h4>Polyphosphate Kinase (PPK) Fusion Protein</h4>
 
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<p class="aim">Basic aim: Assess impact localisation tag has on kinetics of PPKs</p>
 
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<h3><b>Introduction<b></h3><br>
 
<h3><b>Introduction<b></h3><br>
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<p>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.</p>
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<p>This summer, we developed a system which could sequester and store high levels of phosphate: Phosphostore. In our project,
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we expressed Eut (Ethanolamine utilisation) bacterial microcompartments and a polyphosphate kinase (PPK) enzyme with a Pdu (1,2-propanediol utilisation) localization tag. This enables the encapsulation of the PPK enzyme within the microcompartment,
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<p>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.</p>
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allowing the formation of polyphosphate chains to be built and stored safely from degradation by endogenous exopolyphosphatase (PPX). To improve our system, we chose a PPK from <i>Corynebacterium glutamicum</i> as our PPK of choice as it has a 30 fold greater turnover rate than that of the <i>Escherichia coli</i> PPK</p>
 
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Revision as of 17:27, 1 November 2017

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Project Description


Introduction


This summer, we developed a system which could sequester and store high levels of phosphate: Phosphostore. In our project, we expressed Eut (Ethanolamine utilisation) bacterial microcompartments and a polyphosphate kinase (PPK) enzyme with a Pdu (1,2-propanediol utilisation) localization tag. This enables the encapsulation of the PPK enzyme within the microcompartment, allowing the formation of polyphosphate chains to be built and stored safely from degradation by endogenous exopolyphosphatase (PPX). To improve our system, we chose a PPK from Corynebacterium glutamicum as our PPK of choice as it has a 30 fold greater turnover rate than that of the Escherichia coli PPK