Use of mCherryOpt Fluorescent Protein in Clostridium difficile

Here we describe protocols for using the red fluorescent protein mCherryOpt in Clostridium difficile. The protocols can be readily adapted to similar fluorescent proteins (FPs), such as green fluorescent protein (GFP) and cyan fluorescent protein (CFP). There are three critical considerations for using FPs in C. difficile. (1) Choosing the right color: Blue and (especially) red are preferred because C. difficile exhibits considerable yellow-green autofluorescence. (2) Codon optimization: Most FP genes in general circulation have a GC content of ~60 %, so they are not well expressed in low-GC bacteria. (3) Fixing anaerobically grown cells prior to exposure to O2: The FPs under consideration here are non-fluorescent when produced anaerobically because O2 is required to introduce double bonds into the chromophore. Fixation prevents C. difficile cells from becoming degraded during the several hours required for chromophore maturation after cells are exposed to air. Fixation can probably be omitted for studies in which maintaining cellular architecture is not important, such as using mCherryOpt to monitor gene expression.