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Rob Fagan @robfagan.bsky.social · 19/02/2026

To the cloned gene you can add a number of different tags for purification, Western blot detection and fluorescence microscopy - His, HA, StrepTag, mCherry, mScarlet, SNAP, CLIP

Figure 2 from https://doi.org/10.1099/mic.0.001665 - showingWestern blot detection of 3xHA and 6xHis tagged proteins, and purification of a StrepTagged protein

Figure 3 from https://doi.org/10.1099/mic.0.001665 - showing fluorescence microscopy of C. difficile and C. sporogenes expressing a range of red fluorescent fusions

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Rob Fagan @robfagan.bsky.social · 19/02/2026

Finally we generated a set of knock-in plasmids, including 12 with genetic barcodes that allow the C. difficile genome to be tagged with a 9bp sequence that allows unambiguous identification in sequencing datasets

Figure 4 from https://doi.org/10.1099/mic.0.001665 - showing the use of genetic barcodes to distinguish strains in mixed populations during competitive growth in vitro or in the mouse gut

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Rob Fagan @robfagan.bsky.social · 19/02/2026

These can be used for identification of strains in mixed populations - particularly useful for competitive index experiments. You can potentially use 12 times fewer mice in infection studies.

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