Team:Hong Kong HKU/achievements/result

From 2013.igem.org

• E. CAPSI
  • At a Glance
  • M1: Phosphate Removal
  • M2: Polyphosphate Kinase
  • M3: Micro-compartment
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Overview

In order to accomplish our project, we put much energy into our experiment. Here is a summary of what we accomplished in our research over the summer.

Polyphosphate Kinase 1 (ppk1)

The Confirmation Experiment of ppk1 gene




We get the polyphosphate kinase 1 (ppk1) gene from both Kingella oralis (BBa_K1217003) and Tannerella forsythia (BBa_K1217000) and express ppk1 enzyme under the control of T7 promoter (BBa_K525998).

We also fused an N-terminal targeting sequence to the ppk1 gene (BBa_K1217004, BBa_K1217005). This N-terminal targeting sequence is the first 19 amino acid of the Eut C gene from the Salmonella Enterica, which acts as a localizing signal to direct ppk1 enzyme into the Eut Microcompartment (MCP) (BBa_K311004) being assembled in E.capsi.



We express the heterogeneous ppk1 in E.coli BL21 under iptg induction (see protocol) and find out that K. oralis ppk1 shows better expression than T. forsythia ppk1. As shown in the SDS-PAGE analysis, K. oralis ppk1 being expressed is soluble, as well as the Signal fused ppk1. Hence, we choose to use K. oralis ppk1 for the downstream experiment – localizing the ppk1 into the MCP to achieve a higher efficiency of phosphate clearance in the medium.






Eut Microcompartment (MCP)
The Confirmation Experiment of Eut Microcompartment surface decoration.

Localizing ppk1 into MCP and Test for its performance
Since our goal of E. capsi is to enhance the phosphate clearance efficiency from medium by localizing ppk1 enzyme into MCP to accumulate long polyphosphate inside MCP and prevent its degradation by cytosolic ppx enzyme. We want to show E. capsi can (1) uptake greater amount of phosphate from the medium, (2) store higher amount of polyphosphate and (3) accumulate longer polyphosphate chain.
More updated data, please visit: here
Phosphate uptake from the medium (Phosphate Detection Assay)

Polyphosphate concentration inside Bacteria (DAPI assay of polyphosphate)



Length of Polyphosphate Chain inside Bacteria (Toluidine blue staining)