Bsmbi restriction enzyme

A library of DPEase of C. cellulolyticum mutants was generated following the error-prone PCR protocol (35 ) using the OneTaq DNA Polymerase (New England Biolabs), the forward primer 5′-GCCGTCTCGGATGAAACACGGTATCTACTAC-3′, the reverse primer 5′-GCCGTCTCCCGCTTTAAGAGTGTTTGTGGCATTC-3′ and as template a gBlock encoding the C. cellulolyticum DPEase. A control library was performed with the Q5^® High-Fidelity DNA Polymerase (New England Biolabs). Each library was inserted in the Mutant Drop Zone (MDZ) downstream of the psicose biosensor (BBa_K2448057) by Golden Gate, using the BsmBI restriction enzyme (Thermo Fisher Scientific). Ten microliters of the Golden Gate reaction were used to transform chemically competent E. coli DH5α cells. After overnight culturing in LB media supplemented with 35 µg/ml chloramphenicol, transformed cells were centrifuged, washed with IsoFlow Sheath Fluid (Beckman Coulter) and resuspended in this same isotonic fluid at a concentration of 10⁶ cells/ml. Flow cytometric measurements were performed at Genoscope on a MoFlo Astrios cell sorter (Beckam Coulter), using a single laser operating at 488 nm for excitation and a channel of 576/21 nm for detection of the mEmerald fluorescence. The selection was triggered by fluorescence (threshold 0.05%). The data were analyzed using the Summit V6.2 Software (Beckam Coulter).

HG-9-91-01 was synthesized as described elsewhere [2 (link)] and purified to > 96% purity by Syngene International (Bangalore, India). Powder was dissolved in DMSO (Hybri-MAX, Sigma-Aldrich, St. Louis, MO, USA) as 10 mM stock solutions and stored at -20°C until use. Recombinant mouse RANKL was from R&D Systems (Minneapolis, MN, USA). Recombinant mouse M-CSF (rmM-CSF) was from ImmunoTools (Friesoythe, Germany). BSmBI restriction enzyme was from Thermo Fisher Scientific, (Waltham, MA, USA) pLentiCRISPR-V2 (GeCKO), pVSVg (AddGene#8454) and psPAX2 (AddGene #12260) plasmids were a kind gift of Dr. Fabio Martinon (Unil, Lausanne, Switzerland).