B. subtilis strain bGS28, in which the native Pbp2B has been replaced with an IPTG-inducible, Halo-tagged Pbp2B, was imaged as described in Bisson-Filho et al.12 (link). Briefly, cells were grown in CH media, Pbp2B was induced with 20 μM IPTG and labeled with 100 nM JF549 conjugated to HaloTag ligand, and cells were immobilized under an agarose pad for imaging. Cells were imaged in TIRF on a Nikon N-STORM microscope; time lapses were acquired with streaming 30 ms exposures for 1 min. Particle tracking was performed in TrackMate using the simple LAP tracker with the following settings: particle diameter was 300 nm, maximum linking distance was 300 nm, and no frame gaps were allowed. Tracks between 5 and 25 frames were analyzed further using custom MATLAB code12 (link). MSD vs. t was calculated for each track, and the diffusion coefficient was computed by the MSD equation noted above.
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