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

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<h2 id="I">I. Demonstration</h2>
 
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<img class="pictures" id = "21000" src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png"/>
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<p class="pic_text">Fig1.1: CsgA-SpyTag</p>
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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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(Team: Peking, 2016)
 
(Team: Peking, 2016)
 
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<img class="pictures" id = "Improve_1" src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg"/>
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<p class="pic_text">Fig1.2: SpyTag and SpyCatcher System</p>
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<h2 id="ID">II. Verification</h2>
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<h2 id="ID">II. Verification - Using sfGFP-SpyCatcher:</h2>
 
<h3 id="a">CsgA Improvement</h3>
 
<h3 id="a">CsgA Improvement</h3>
 
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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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We used sfGFP-SpyCatcher as indicater to measure the amount of sfGFP-SpyCatcher protein our CsgA-SpyTag biofilm could capture.
 
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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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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#SSS">
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Protocol for SpyTag-SpyCatcher system verification
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The decrease of florescence of the supernatant indicates how much sfGFP-SpyCatcher protein the biofilm gain.
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<img class="pictures" id = "Improve_2" src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png"/>
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There’s a significant difference between the two groups. Thus, our CgsA-SpyTag successfully fixed sfGFP-CatCher proetin.
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<h2 id="ID">III. Verification - Using SpyCatcher-CotA:</h2>
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We used SpyCatcher-CotA Protein, laccase with SpyCatcher, to fix on to our CsgA-SpyTag biofilm. We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole. If our CsgA-SpyTag biofilm has laccase activity, that means the fixing process of SpyCatcher and SpyTag is successful.
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<a href="https://2018.igem.org/Team:SHSBNU_China/Protocol#BXL">
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Protocol for Biofilm x Laccase
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<img class="pictures" id = "Improve_3" src="https://static.igem.org/mediawiki/2018/7/7c/T--SHSBNU_China--Improve_3.png"/>
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There were significance of difference was indicated as **** which was pretty high. So we proved that SpyCatcher-CotA is successfully combined to the biofilm.
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<strong>再整个小标题</strong>
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<h2 id="ID">IV. Conclusion</h2>
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In our experiments, we give CsgA a new ability, which is capturing all fusion protein with SpyCatcher, by adding SpyTag domain after CsgA. We test SpyTag using sfGFP-SpyCatcher and SpyCatcher-CotA. We also proved that fusion protein is still functional after fixed to the biofilm.
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Revision as of 03:15, 18 October 2018

Improvements

Improve

I. Demonstration

Fig1.1: CsgA-SpyTag

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)

Fig1.2: SpyTag and SpyCatcher System

II. Verification - Using sfGFP-SpyCatcher:

CsgA Improvement

We used sfGFP-SpyCatcher as indicater to measure the amount of sfGFP-SpyCatcher protein our CsgA-SpyTag biofilm could capture.

Protocol for SpyTag-SpyCatcher system verification

The decrease of florescence of the supernatant indicates how much sfGFP-SpyCatcher protein the biofilm gain.

There’s a significant difference between the two groups. Thus, our CgsA-SpyTag successfully fixed sfGFP-CatCher proetin.

III. Verification - Using SpyCatcher-CotA:

We used SpyCatcher-CotA Protein, laccase with SpyCatcher, to fix on to our CsgA-SpyTag biofilm. We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole. If our CsgA-SpyTag biofilm has laccase activity, that means the fixing process of SpyCatcher and SpyTag is successful.

Protocol for Biofilm x Laccase

There were significance of difference was indicated as **** which was pretty high. So we proved that SpyCatcher-CotA is successfully combined to the biofilm.

再整个小标题

IV. Conclusion

In our experiments, we give CsgA a new ability, which is capturing all fusion protein with SpyCatcher, by adding SpyTag domain after CsgA. We test SpyTag using sfGFP-SpyCatcher and SpyCatcher-CotA. We also proved that fusion protein is still functional after fixed to the biofilm.

Reference:

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