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Team:Hong Kong HKU/achievements/result
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217016">BBa_K1217016</a>, | <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217016">BBa_K1217016</a>, | ||
<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217017">BBa_K1217017</a>. <br><br> | <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217017">BBa_K1217017</a>. <br><br> | ||
| - | EutS gene is amplified from the BBa_K311004 (Eut SMNLK MCP), Histidine tag and linker is added to the EutS N terminus through PCR. EutS expression is shown in SDS-PAGE and Histidine tag is detected in western blot analysis. Histidine tag EutSMNLK has also been constructed.</font><br> | + | EutS gene is amplified from the <a href="http://parts.igem.org/Part:BBa_K311004">BBa_K311004</a>(Eut SMNLK MCP), Histidine tag and linker is added to the EutS N terminus through PCR. EutS expression is shown in SDS-PAGE and Histidine tag is detected in western blot analysis. Histidine tag EutSMNLK has also been constructed.</font><br> |
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| + | <img src="https://static.igem.org/mediawiki/parts/6/6f/HKU13_HisEutSWestern.png" height="250" width=auto><br><br> | ||
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Revision as of 01:47, 28 September 2013
Result
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.
For more updates, please check our Biobrick Registry! BBa_K1217003, BBa_K1217008, BBa_K1217015.
Polyphosphate Kinase 1 (ppk1)
Construction of PPK1
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).
SDS-PAGE analysis of K. oralis ppk1 shows strong and soluble expression while T. forsythia ppk1 expression does not show distinguishable difference compared with control(Figure below).
After confirming the expression of native PPK1. We fused an N-terminal targeting sequence with 19 amino acids 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. Both signal-PPK1 expression has been detected. Since native K. oralis PPK1 shows better expression, we 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 Assembly and Surface Tagging
Construction of EutS MCP, Eut SMNLK MCP and their His-tagged version
BBa_K1217015,
BBa_K1217016,
BBa_K1217017.
EutS gene is amplified from the BBa_K311004(Eut SMNLK MCP), Histidine tag and linker is added to the EutS N terminus through PCR. EutS expression is shown in SDS-PAGE and Histidine tag is detected in western blot analysis. Histidine tag EutSMNLK has also been constructed.
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)
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