Difference between revisions of "Part:BBa K2684006"
 
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<p>Fig1.1: CsgA-SpyTag<br>
 
<p>Fig1.1: CsgA-SpyTag<br>
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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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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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<h2>Verification- Using sfGFP-SpyCatche</h2>
 
<h2>Verification- Using sfGFP-SpyCatche</h2>
 
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<p>
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We used sfGFP-SpyCatcher as indicator to measure the amount of sfGFP-SpyCatcher protein our CsgA-SpyTag biofilm could capture. The decrease of florescence of the supernatant indicates how much sfGFP-SpyCatcher protein the biofilm gain.
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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:75%">
 
<img src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png" style="width:75%">
 
<p>
 
<p>
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There’s a significant difference between the two groups. Thus, our CsgA-SpyTag successfully fixed sfGFP-CatCher protein.
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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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</p>
 
<h2>Verification- Using SpyCatcher-CotA</h2>
 
<h2>Verification- Using SpyCatcher-CotA</h2>
 
<p>
 
<p>
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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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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%">
 
<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 CsgA-Spytag in enzyme activity when combining with SpyCatcher-CotA<br>
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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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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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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.