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4 protocols using bsmbi restriction enzyme

1

Mutagenesis and Screening of C. cellulolyticum DPEase

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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).
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2

Recombinant Protein Production and CRISPR Plasmids

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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).
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Constructing pLentiCRISPR-CARM1 Plasmid

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pLentiCRISPR-CARM1 was constructed by inserting the CARM1 guide RNA (gRNA; 5′-AGCACGGAAAATCTACGCGG-3′). pLentiCRISPR v2 (Addgene) was digested and dephosphorylated with BsmBI restriction enzyme (Fermentas) for 30 min at 37 °C. The digested plasmid was run on a 1% agarose gel, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and then were ramped down to 25 °C at 5 °C/min. Annealed oligonucleotides were diluted 1:20 0 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested pLentiCRISPR v2 plasmid was performed using Quick Ligase (New England Biolabs, Inc.).
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4

CARM1 Overexpression via pLentiCRISPR

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pLentiCRISPR-CARM1 was constructed by inserting the CARM1 guide RNA (gRNA; 5′-AGCACGGAAAATCTACGCGG-3′)35 (link). pLentiCRISPR v2 (Addgene) was digested and dephosphorylated with BsmBI restriction enzyme (Fermentas) for 30 min at 37 °C. The digested plasmid was run on a 1% agarose gel, cut out, and purified using the Wizard SV Gel and PCR Clean Up kit (Promega). The oligonucleotides were phosphorylated using T4 PNK (M0201S) with T4 Ligation Buffer (New England Biolabs, Inc.). Samples were annealed in a thermocycler at 37 °C for 30 min and then at 95 °C for 5 min and then were ramped down to 25 °C at 5 °C/min. Annealed oligonucleotides were diluted 1:200 in RNase/DNase-free water. Ligation of the annealed oligonucleotide and digested pLentiCRISPR v2 plasmid was performed using Quick Ligase (New England Biolabs, Inc.).
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