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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<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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<h3 id="a">CsgA Improvement</h3>
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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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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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The decrease of florescence of the supernatant indicates how much sfGFP-SpyCatcher protein the biofilm gain.
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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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There’s a significant difference between the two groups. Thus, our CgsA-SpyTag successfully fixed sfGFP-CatCher proetin.
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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="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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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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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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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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<strong>再整个小标题</strong>
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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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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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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.