Difference between revisions of "Team:SHSBNU China/Improve"

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<h6 id="menu_intro">Improve</h6>
 
<h6 id="menu_intro">Improve</h6>
 
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#I">Overview</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#I">Demonstration</a>
 
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#ID">Improvement Details</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#ID">Verification</a>
 
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            <a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#a">CsgA Improvement</a>
 
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            <a class="trd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#b">CotA Improvement</a>
 
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<h2 id="I">I. Overview</h2>
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<h2 id="I">I. Demonstration</h2>
 
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<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:100%"/></image>
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<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:100%">
 
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    Previously, there was only CsgA part uploaded in iGEM parts website. Our Team improved part <a href="http://parts.igem.org/Part:BBa_K1583000">Part BBa_K1583000</a> by adding a SpyTag sequence. It fused to gene <em>csgA</em>, enabling CotA laccase to be fixed onto the biofilm to form a covalent bond--SpyTag-SpyCatcher.  
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    CsgA is a kind of amyloid protein that could form biofilm in E. coli cells. We fused SpyTag domain after gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system and the biofilm forming amyloid protein, it is possible to fix any SpyChatcher fused protein domains to the biofilm.  
 
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(Team: Peking, 2016)
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<img src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg" style="width:100%"/></image>
 
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<h2 id="ID">II. Improvement Details</h2>
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<h2 id="ID">II. Verification</h2>
 
<h3 id="a">CsgA Improvement</h3>
 
<h3 id="a">CsgA Improvement</h3>
 
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By using sfGFP-SpyCatcher protein, we tested the binding efficiency of the covalence SpyTag-SpyCatcher since sfGFP is a non-toxic and common used flurorescent protein. Gene <em>csgA</em> on the plasmid of pET28a was transferred into ΔMG1655 as control group. Gene <em>csgA – spytag</em> on the plasmid of pET28a was transferred into ΔMG1655 as experiment. The experiment was divided into 4 groups. The bateria in the Reaction Stock had neither <em>csgA</em> nor <em>SpyTag</em> in neither genome nor plasmid; the ones in Group 1 had <em>csgA</em> in the genome; the ones in Group 2 had <em>sfGFP</em> in the plasmid; the ones in Group 3 had <em>csgA-SpyTag</em> in the genome and the plasmid. The treatment of the experiment is followed by the procedure below:
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CsgA and CsgA-SpyTag were both cloned into the plasmid of pET28a for further improvement assay. We transferred both part in to ΔCsgA-MG1655 cells as two groups and incubate them till OD≈0.6. Both groups were centrifuged. We used a reaction stock containing SpyCatcher-sfGFP protein, of which the florescence was measured, to react with the palette of the two groups. We allow the reaction undergo 1 hours. After that, we centrifuged both groups again and measure the florescence of the supernatant. The amount of florescence the supernatant decreased indicates how much SpyCatcher-sfGFP protein the palette fixed.
 
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<img src="https://static.igem.org/mediawiki/2018/9/92/T--SHSBNU_China--improve1.jpg" style="width:100%">
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<img src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png" style="width:100%">
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<img class="pictures" id = "21002" src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg"/>
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<p class="pic_text">The liquid after centrifugation was extracted and measured fluorescence. </p>
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  As can be seen from the result, the experiment group showed the most decrease of sfGFP-SpyCatcher protein. The difference between the control group and experiment group was significant. Thus we could confirm our CsgA-SpyTag system was functional.
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There’s a significant difference between the two groups. Thus, our CgsA-SpyTag is functional whenconbining with proetin fused wity SpyCatcher domain.
 
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                      Part BBa_K2684000 was improved from previous part <a href="http://parts.igem.org/Part:BBa_K1336002">BBa_K1336002</a>. Our team made a point mutation to eliminate the EcoR1 cutting site so that we can meet the standard of RFC10, a commonly-used standard for interchangeable parts which are based on idempotent assembly. We tested the enzyme activity of our part based on the weight of our sample. Formula: laccase activity (nmol/min/g) = ΔA /ε(ABTS mmolar extinction coefficient) / d * V (total volume of reaction) / V (volume of sample in the reaction, 0.025mL) / W (sample mass, g) * V (extracted liquid added, 1mL) / T (reaction time, 3 minutes) = 130 *ΔA / W. The result is shown as following:
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<strong>Reference:</strong>
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                    <img src="https://static.igem.org/mediawiki/2018/4/42/T--SHSBNU_China--Parts_Regis.png" style="width:100%"/>
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“Uranium Reaper.” <i>Uranium Reaper - Team: Peking</i>, 2016.igem.org/Team:Peking.
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                      The improvement is valid since it was normally produced.
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Revision as of 02:49, 18 October 2018

Improvements

Improve

I. Demonstration

CsgA is a kind of amyloid protein that could form biofilm in E. coli cells. We fused SpyTag domain after gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system and the biofilm forming amyloid protein, it is possible to fix any SpyChatcher fused protein domains to the biofilm.

(Team: Peking, 2016)

II. Verification

CsgA Improvement

CsgA and CsgA-SpyTag were both cloned into the plasmid of pET28a for further improvement assay. We transferred both part in to ΔCsgA-MG1655 cells as two groups and incubate them till OD≈0.6. Both groups were centrifuged. We used a reaction stock containing SpyCatcher-sfGFP protein, of which the florescence was measured, to react with the palette of the two groups. We allow the reaction undergo 1 hours. After that, we centrifuged both groups again and measure the florescence of the supernatant. The amount of florescence the supernatant decreased indicates how much SpyCatcher-sfGFP protein the palette fixed.

There’s a significant difference between the two groups. Thus, our CgsA-SpyTag is functional whenconbining with proetin fused wity SpyCatcher domain.

Reference:

“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.