This plasmid was transformed into T7 Express lysY/Iq competent Escherichia coli (New England BioLabs). A 0.1% inoculum was used, and cell cultures were incubated aerobically at 37 °C with vigorous shaking. When the optical density (600 nm) reached a value of 0.6, the incubation temperature was reduced to 30 °C, and expression of the fusion protein was induced with 0.1 mM IPTG for 15 min. Cells were collected by centrifugation for 30 min at 6000 g, supernatant was discarded,

and pellets were frozen overnight. Cell pellets were resuspended 10× in Lysis Buffer containing 25 mM Tris–HCl, pH 7.4, Fulvestrant datasheet 250 mM NaCl, 8 M Urea. A final volume of 3 mL was sonicated for 5 min total process time (30 s on, 30 s off) using Misonix S-4000 (Misonix Inc.) with the amplitude set to 55%. Cell debris was removed by centrifugation for 30 min at 6000 g, and the supernatant containing the fusion protein was collected for further analysis. Supernatant was dialyzed to Dockerin Reaction Buffer

(25 mM Tris–HCl, pH 7.4, 50 mM NaCl, 1 mM CaCl2, 1 mM DTT, 0.1% Tween 20). The sample was centrifuged for 30 min at 6000 g to remove precipitates formed during dialysis, and pellet was discarded. Supernatant BIRB 796 mouse containing the SNAP-XDocII fusion protein in Dockerin Reaction Buffer was used in all subsequent labeling experiments. Expression of the SNAP-XDocII fusion protein was optimized to include a short induction period of 15 min at 30 °C. Protocols for recovery of the SNAP-XDocII fusion protein were adapted from Adams et al. (2004). Under these conditions, the soluble SNAP-XDocII fusion protein was recovered at a final concentration of 2.5 mM. The SNAP-XDocII fusion protein exhibited covalent

binding to the SNAP fluorophore, as determined by SDS-PAGE analysis. Optimized parameters for labeling the fusion protein with SNAP fluorophore resulted in complete labeling of the fusion protein, with unbound fluorophore remaining in solution at < 50% of the concentration of the fusion protein. Fusion proteins for flow cytometry and microscopy were labeled with ifoxetine SNAP-Surface® Alexa Fluor® 647 and SNAP-Cell® 505 fluorescent dyes (New England BioLabs) by incubation of 2.5 mM fluorescent dye with fusion protein at 37 °C for 1 h. The resulting fluorescent proteins are referred to as 505-SNAP-XDocII and 647-SNAP-XDocII (Fig. S1). Before incubation with C. thermocellum, the labeling reaction was centrifuged to remove nonfluorescent precipitates that formed during 37 °C incubation. For fluorescent SDS-PAGE analysis, fusion protein was labeled with SNAP-Vista® Green according to the manufacturer’s instructions (New England BioLabs). Volumes of C. thermocellum culture, grown to an OD600 nm of 0.5 were harvested by centrifugation for 2 min at 15 000 g. Cell pellets were resuspended with an equal volume of 0.