Difference between revisions of "Part:BBa K2684006"
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===Usage and Biology===
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===Functional Parameters===
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<h2>Usage and Biology</h2>
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<h2>Demonstration</h2>
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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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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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<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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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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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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(Team: Peking, 2016)
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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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<h2>Demonstration</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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<img src="https://static.igem.org/mediawiki/2018/9/90/T--SHSBNU_China--Improve_2.png" style="width:100%">
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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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<strong>Reference:</strong>
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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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Revision as of 02:48, 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

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)

Demonstration

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.

There’s a significant difference between the two groups. Thus, our CgsA-SpyTag is functional whenconbining with proetin fused wity SpyCatcher domain.

Reference:

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