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

 
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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="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VI">Using sfGFP-SpyCatcher</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#VII">Verification - Using SpyCatcher-CotA</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Improve#C">Conclusion</a>
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<a class="snd_class" href="https://2018.igem.org/Team:SHSBNU_China/Project">Project</a>
 
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<h2 id="I">I. Demonstration</h2>
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<h2 id="I">I. Overview</h2>
 
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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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<div style="width: 46vw" class="content_pic_left">
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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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    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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    CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with 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 , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.  
 
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(Team: Peking, 2016)
 
(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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<div style="width: 46vw" class="content_pic_left">
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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="VI">II. Verification - Using sfGFP-SpyCatcher</h2>
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<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 indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins. </p>
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<p class="text">
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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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</a>
 
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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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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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We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).</p>
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<div style="width: 46vw" class="content_pic_left">
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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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<p class="pic_text"></p>
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The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.</p>
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<h2 id="VII">III. Verification - Using SpyCatcher-CotA</h2>
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<div class="content">
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<div style="width: 20vw" class="content_pic_left">
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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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<p class="pic_text"></p>
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<p class="text">
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The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity. </p>
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<p class="text">
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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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</a>
 
</p>
 
</p>
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<p class="text">
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The difference between our improved part and the previous part is significant.  Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag. </p>
  
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<br>
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<h2 id="C">IV. Conclusion</h2>
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To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.
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<p style="text-indent:0" class="text">
 
<strong>Reference:</strong>
 
<strong>Reference:</strong>
 
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Latest revision as of 03:55, 18 October 2018

Improvements

Improve

I. Overview

Fig1.1: CsgA-SpyTag

CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with 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 , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.

(Team: Peking, 2016)

Fig1.2: SpyTag and SpyCatcher System

II. Verification - Using sfGFP-SpyCatcher

We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins.

Protocol for SpyTag-SpyCatcher system verification

We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).

The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.

III. Verification - Using SpyCatcher-CotA

The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity.

Protocol for Biofilm x Laccase

The difference between our improved part and the previous part is significant. Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag.



IV. Conclusion

To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.

Reference:

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