Difference between revisions of "Part:BBa K2684000"
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<h2>Period - <i>CsgA - SpyTag</i></h2>
 
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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. A <i>Spytag</i> sequence was then fused after <i>csgA</i> gene, creating <i>csgA-spycatcher</i> (BBa_K2684006).  
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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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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><img src="https://static.igem.org/mediawiki/2018/8/87/T--SHSBNU_China--21001.jpg" style="width:50%"/></image></p>
 
<p><img src="https://static.igem.org/mediawiki/2018/8/87/T--SHSBNU_China--21001.jpg" style="width:50%"/></image></p>
 
<p class="pic_text">Reaction stock leftover in experiment</p>
 
<p class="pic_text">Reaction stock leftover in experiment</p>

Revision as of 11:21, 17 October 2018

       CotA Laccase of B.subtilis
       CotA Laccase is an endospore type protein secreted from B.subtilis

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1348
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 286

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.

Reaction stock leftover in experiment

Using sfGFP – spycatcher protein, the combing function of Spytag and spycatcher system on the biofilm was tested. Gene csgA on the plasmid of pET28a was transferred in to ΔMG1655 as control group. Gene csgA – Spytag on the plasmid of pET28a was transferred in to ΔMG1655 as experiment. To verify the function of Spytag on csgA, 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 SpyTag-SpyCatcher system verification

As can be seen from the result,

Thus we can confirm our csgA – SpyTag system is functional.

References

Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.