Difference between revisions of "Part:BBa K2684006"
 
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CsgA fused with SpyTag by 2xGGGGS linker
 
CsgA fused with SpyTag by 2xGGGGS linker
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<br>
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This part was improved from BBa_K1583000: https://parts.igem.org/Part:BBa_K1583000
  
 
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===Usage and Biology===
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===Usage and Biology===-->
  
 
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===Functional Parameters===
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===Functional Parameters===-->
 
<partinfo>BBa_K2684006 parameters</partinfo>
 
<partinfo>BBa_K2684006 parameters</partinfo>
 
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<h2>Period - <i>CsgA - SpyTag</i></h2>
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<h2>Demonstration</h2>
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<p><img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:75%"/></image></p>
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<img src="https://static.igem.org/mediawiki/2018/b/b9/T--SHSBNU_China--21000.png" style="width:75%"/></image>
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Gene <i>csgA</i> found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene <i>csgA</i> on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene <i>csgA</i> was fused into plasmid pET28a.</p>  
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<p>Fig1.1: CsgA-SpyTag<br>
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The CsgA sequence was improved from <a href="https://parts.igem.org/Part:BBa_K1583000">Part BBa_K1583000</a>. We added a SpyTag sequence which fused after <i>csgA</i> gene, creating <i>csgA-spytag</i> (BBa_K2684006). With SpyTag, CotA laccase can be fixed onto the biofilm by forming a covalent bond SpyTag-SpyCatcher.</p>
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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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<p><img src="https://static.igem.org/mediawiki/2018/8/87/T--SHSBNU_China--21001.jpg" style="width:50%"/></image></p>
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</p>
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<p class="pic_text">Reaction stock leftover in experiment</p>
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<img src="https://static.igem.org/mediawiki/2018/b/bb/T--SHSBNU_China--Improve_1.jpg" style="width:75%"/></image>
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</div>
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<p>
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<p class="text">
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Fig1.2: SpyTag and SpyCatcher System
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Using sfGFP(<a href="https://parts.igem.org/Part:BBa_K2684004">part: BBa_K2684004</a>) – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene <i>csgA</i> on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene <i>csgA – Spytag</i> on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on <i>csgA</i>, the experiment was design to compare the combing rate of sf-GFP – spycatcher protein with cells that have csgA – SpyTag (Experiment) or csgA (Control).  
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（Source From Peking 2016 wiki）
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</p>
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<h2>Verification- Using sfGFP-SpyCatche</h2>
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<p>
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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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</p>
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<img src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png" style="width:75%">
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<p>
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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>
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<h2>Verification- Using SpyCatcher-CotA</h2>
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<p>
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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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</p>
 
</p>
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<p><img src="https://static.igem.org/mediawiki/2018/5/57/T--SHSBNU_China--21002.jpg" style="width:50%"/></image></p>
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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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Link: Protocol for <a href="http://2018.igem.org/Team:SHSBNU_China/Protocal#SSS">SpyTag-SpyCatcher</a> system verification
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<p>
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</p>
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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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<p class="text">
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As can be seen from the result,  
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</p>
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<p class="text">
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Thus we can confirm our <i>csgA – SpyTag</i> system is functional.
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</p>
 
</p>
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<p>
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<strong>Reference:</strong>
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</p>
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<p>
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“Uranium Reaper.” <i>Uranium Reaper - Team: Peking</i>, 2016.igem.org/Team:Peking.
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</body>
 
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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.