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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<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.  
 
    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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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.
 
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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There were significance of difference was indicated as **** which was pretty high. So we proved that SpyCatcher-CotA is successfully combined to the biofilm.
 
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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Revision as of 03:18, 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.