Team:Hong Kong HKU/achievements/result

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<p><font face="Impact" size="7" color="green">
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Overview
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Results</font><br>
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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.<br>
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For more updates, please check our Biobrick Registry!  <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217003">BBa_K1217003</a>,  <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217008">BBa_K1217008</a>,  <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217015">BBa_K1217015</a>.
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Polyphosphate Kinase 1 (ppk1)</font>
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<font face="century gothic" size="3" color="Purple"><b>Construction of PPK1</b><br><br>
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<img src="https://static.igem.org/mediawiki/parts/c/c3/Biobrickppk.png" height="80" width=auto><br>
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We get the polyphosphate kinase 1 (ppk1) gene from both Kingella oralis (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217003">BBa_K1217003</a>) and Tannerella forsythia ( <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217000">BBa_K1217000</a>) and express ppk1 enzyme under the control of T7 promoter ( <a href="http://parts.igem.org/Part:BBa_K525998">BBa_K525998</a>). <br><br>
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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).</font>
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<img src="https://static.igem.org/mediawiki/parts/5/51/KOTFppkSDSPAGE.png" height="350" width=auto><br><br>
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After confirming the expression of native PPK1. We fused an N-terminal targeting sequence with 19 amino acids to the ppk1 gene (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217004">BBa_K1217004</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217005">BBa_K1217005</a>). 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) ( <a href="http://parts.igem.org/Part:BBa_K311004">BBa_K311004</a>) 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.
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<img src="https://static.igem.org/mediawiki/parts/2/2c/Biobricksigppk.png" height="80" width=auto><br><br>
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<img src="https://static.igem.org/mediawiki/parts/5/57/KOTFsigppk.png" height="350" width=auto><br><br>
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<br>Eut Microcompartment Assembly and Surface Tagging<br><br>
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<b>Construction of EutS MCP, Eut SMNLK MCP and their His-tagged version</b>
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217015">BBa_K1217015</a>,
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217016">BBa_K1217016</a>,
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<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1217017">BBa_K1217017</a>. <br><br>
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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/8/8a/HKU13_EutSHisEutS_sdspage.png" height="300" width=auto>
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<img src="https://static.igem.org/mediawiki/parts/6/6f/HKU13_HisEutSWestern.png" height="200" width=auto><br><br>
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<br>Localizing ppk1 into MCP and Test for its performance<br><br>
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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.
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All samples, BL21 (control), PPK1, Signal-PPK1+EutS MCP and Signal-PPK1+EutSMNLK MCP were incubate at 16 °C in 0.5mM IPTG for 16 hours. Medium and cell pellet samples in end point are collect to measure the medium phosphate levels and intracellular polyP levels.
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More updated data, please visit: <a href="http://parts.igem.org/Part:BBa_K1217008">here</a>
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<br><b>Phosphate uptake from the medium (Phosphate Detection Assay)</b></font><br><br>
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<font face="century gothic" size="3">We use BIOMOL GREENTM reagent kit to measure the phosphate concentration of the medium. It is a modification of the classic Malachite green reagent and offers excellent sensitivity.<br><br></font>
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<font face="century gothic" size="3", color="purple">
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<b>Polyphosphate concentration inside Bacteria (DAPI assay of polyphosphate)</b><br></font>
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<br><font face="century gothic" size="3">Cell pellet is used for extracting cellular polyP and the sample amount is normalized by protein concentration using Bradford Assay.<br><br>
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PolyP amount is quantified using DAPI fluorescent assay. The experiment has been performed only once and we are trying to replicate this experiment to gather statistical meaningful results. Based on this single result, it is promising as over-expression of PPK1 contains double amount of PolyP compared to wild type control.<br>
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<img src=" https://static.igem.org/mediawiki/2013/e/e9/WholeDAPIassay1.png" height="300" width=auto><br><br>
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<font face="century gothic" size="3", color="purple">
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<br><b>Length of Polyphosphate Chain inside Bacteria (Toluidine blue staining)</b><br></font>
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<br><font face="century gothic" size="3">Cellular PolyP concentrations is further visualized by the loading the PolyP extract to urea gel using Toluidine blue staining. Normalized amount of samples were loaded in lane as indicated and lane 5 acts as a positive control which contain 1 μg of polyP. Difference of PolyP amount between samples cannot be distinguished by colour difference since the polyP amount might be over saturated as compare to the control.<br></font>
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<img src="https://static.igem.org/mediawiki/2013/4/48/Polypgel1.jpg" height="350" width=auto><br><br>
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Latest revision as of 03:32, 28 September 2013


Achievements

Biobricks Results Judging Criteria
Back to Top



Results

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. All samples, BL21 (control), PPK1, Signal-PPK1+EutS MCP and Signal-PPK1+EutSMNLK MCP were incubate at 16 °C in 0.5mM IPTG for 16 hours. Medium and cell pellet samples in end point are collect to measure the medium phosphate levels and intracellular polyP levels.
More updated data, please visit: here

Phosphate uptake from the medium (Phosphate Detection Assay)

We use BIOMOL GREENTM reagent kit to measure the phosphate concentration of the medium. It is a modification of the classic Malachite green reagent and offers excellent sensitivity.

 Polyphosphate concentration inside Bacteria (DAPI assay of polyphosphate)
Cell pellet is used for extracting cellular polyP and the sample amount is normalized by protein concentration using Bradford Assay.

PolyP amount is quantified using DAPI fluorescent assay. The experiment has been performed only once and we are trying to replicate this experiment to gather statistical meaningful results. Based on this single result, it is promising as over-expression of PPK1 contains double amount of PolyP compared to wild type control.


Length of Polyphosphate Chain inside Bacteria (Toluidine blue staining)
Cellular PolyP concentrations is further visualized by the loading the PolyP extract to urea gel using Toluidine blue staining. Normalized amount of samples were loaded in lane as indicated and lane 5 acts as a positive control which contain 1 μg of polyP. Difference of PolyP amount between samples cannot be distinguished by colour difference since the polyP amount might be over saturated as compare to the control.



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