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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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>
<p class="pic_text">Reaction stock leftover in experiment</p>
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Using sfGFP – 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).
