<p class="pic_text">Reaction stock leftover in experiment</p>
<p class="pic_text">Reaction stock leftover in experiment</p>
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Using sfGFP–spycatcher protein(<a href="https://parts.igem.org/Part:BBa_K2684004">Part: BBa_K2684004</a>), 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).
Link: Protocol for <a href="http://2018.igem.org/Team:SHSBNU_China/Protocal#SSS">SpyTag-SpyCatcher</a> system verification
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As can be seen from the result,
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Thus we can confirm our <i>csgA – SpyTag</i> system is functional.
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Revision as of 12:03, 17 October 2018
CsgA-SpyTag
CsgA fused with SpyTag by 2xGGGGS linker
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]
Period - CsgA - SpyTag
Gene csgA found in the genome of MG1655 wild type is capable of forming biofilm. Using CRISPR, we knocked out gene csgA on MG1655’s genome creating ΔMG1655 strain. The cell ΔMG1655 would then be used as chassis cell. Gene csgA was fused into plasmid pET28a.
The CsgA sequence was improved from Part BBa_K1583000. We added a SpyTag sequence which fused after csgA gene, creating csgA-spytag (BBa_K2684006). With SpyTag, CotA laccase can be fixed onto the biofilm by forming a covalent bond SpyTag-SpyCatcher.
