Part:BBa_K3183300

ProQuorum C. difficile Detection and CD27L Secretion System

This part enables L. reuteri cells to detect C. difficile quorum signalling molecules and secrete a specific endolysin derived from phage CD27L to lyse C. difficile cells. This part, if functional, would be a demonstration of our system's capabilities to detect and lyse C. difficile. This part also includes a SpyTag and His tag, both codon optimised for L. reuteri, to allow for CD27L endolysin purification.

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]

Future Experiments (2019)

The Oxford 2019 iGEM team attempted to characterise all the component parts within this system. CD27L was well characterised against B. subtilis and constructs were made for the Agr-sensing system. However, we were unable to detect expression of AgrA and AgrC (BBa_K3183102), which prevented further work with the putative promoter.

Finding and confirming the AgrAC putative promoter BBa_K3183003 would require a number of experiments. Phosphorylated and/or dimerised AgrA could be fixed to a column using a tag (e.g. SpyTag) and DNA pull-downs could be performed following genomic DNA shearing. This could show potential DNA binding regions which haven’t been proven from previous gene-expression experiments⁴. Further confirmation could be obtained from footprinting, electrophoretic mobility shift assays (EMSA), or nitrocellulose filter assays.

Ultimately, the goal would to show that a fully functional signalling system can be integrated in the cell biology of Lactobacillus and that expression of the endolysin can be induced by the binding of C. difficile AIP to the AgrC receptor. This could be tested using a fluorescence reporter assay.