Team:Hong Kong HKU/achievements/result

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. Latest data will be updated into the Biobrick Resgitry: Samuel, display the biobrick Table here. ONLY display the Following BIObirck in the table. Others are useless and I don’t wanna cause distractions. BBa_K1217000 BBa_K1217003 BBa_K1217004 BBa_K1217005 BBa_K1217008 BBa_K1217009 BBa_K1217010 BBa_K1217011 BBa_K1217015 BBa_K1217016 BBa_K1217017 BBa_K1217019 BBa_K1217020 BBa_K1217021 BBa_K1217022 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). biobrickppk.png 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. biobricksigppk 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. HKU13_KOppkSDS.png KO,KOsigppk.png 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: http://parts.igem.org/Part:BBa_K1217008 Phosphate uptake from the medium (Phosphate Detection Assay) Polyphosphate concentration inside Bacteria (DAPI assay of polyphosphate) DAPIexp1.png Length of Polyphosphate Chain inside Bacteria (Toluidine blue staining) [File:polyp_gel_trail_1.png/200px/thumb/left/alt polypgel]]