Difference between revisions of "Part:BBa K2684006"
 
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<h2>Demonstration</h2>
 
<h2>Demonstration</h2>
 
<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:75%"/></image>
 
<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:75%"/></image>
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<p>Fig1.1: CsgA-SpyTag<br>
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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 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)
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</p>
 
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg" style="width:75%"/></image>
 
<img src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg" style="width:75%"/></image>
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<h2>Demonstration</h2>
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Fig1.2: SpyTag and SpyCatcher System
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（Source From Peking 2016 wiki）
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<h2>Verification- Using sfGFP-SpyCatche</h2>
 
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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.
 
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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 src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png" style="width:75%">
 
<p>
 
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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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There’s a significant difference between the two groups. This suggests our CsgA-SpyTag could sufficiently connect with SpyCatcher-CotA .
 
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<h2>Verification- Using SpyCatcher-CotA</h2>
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We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole.
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<img src="https://static.igem.org/mediawiki/2018/4/4a/T--SHSBNU_China--23000.jpg" style="width:75%">
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As the **** shows, there is significant difference between CsgA and SpyTag-CsgA in enzyme activity when combining with SpyCatcher-CotA<br>
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<strong>Reference:</strong>
 
<strong>Reference:</strong>
 
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Latest revision as of 03:47, 18 October 2018

CsgA-SpyTag

CsgA fused with SpyTag by 2xGGGGS linker
This part was improved from BBa_K1583000: https://parts.igem.org/Part:BBa_K1583000


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Demonstration

Fig1.1: CsgA-SpyTag
CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused 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.

Fig1.2: SpyTag and SpyCatcher System （Source From Peking 2016 wiki）

Verification- Using sfGFP-SpyCatche

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.

There’s a significant difference between the two groups. This suggests our CsgA-SpyTag could sufficiently connect with SpyCatcher-CotA .

Verification- Using SpyCatcher-CotA

We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole.

As the **** shows, there is significant difference between CsgA and SpyTag-CsgA in enzyme activity when combining with SpyCatcher-CotA

Reference:

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