Part:BBa_K3183104

Putative Agr Promoter for C. difficile Fused with mClover3

This part contains the putative Agr promoter (BBa_K3183003which we believe is activated by the AgrAC two component signalling system. To test the effectiveness and strength of the promoter, we fused it to reporter mClover3 (BBa_K3183010), so that the promoter function can be determined through the detection of fluorescence.

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 the AgrAC system before attempting to work with the putative promoter. However, we were unable to detect expression of AgrA or 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.