E coli bl21

All E. coli strains were maintained in Luria broth (LB) medium at 37 °C with appropriate antibiotics and/or supplements unless stated otherwise (final concentrations: 30 mg/L kanamycin and 50 mg/L d-alanine). Cloning was performed with E. coli Top10 (Invitrogen), heterologous protein expression was performed with E. coli BL21 (Thermo Fisher Scientific), and characterisation was performed with a modified E. coli Nissle 1917 EcN strain^{22 (link)}. C. difficile culture was carried out in a Coy Lab Vinyl Anaerobic Chamber and anaerobic jar to maintain anaerobic conditions. Cells were cultured in brain heart infusion-supplemented (BHIS) medium supplemented with 5% w/v yeast extract and 0.03% w/vl-cysteine. Taurocholate was supplemented as necessary. The cells were incubated anaerobically at 37 °C. C. difficile CD630, VPI10463, BAA1870 and 9689 were obtained from the American Type Culture Collection (ATCC). The Caco-2 cell line (ATCC) was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% foetal bovine serum and 1% penicillin/streptomycin (10000 U/mL). The cells were incubated at 37 °C under a 5% CO₂/95% air atmosphere. Cell passage was performed at ~70 to 90% confluence.

HOK-16B cells (RRID: CVCL_B405) were maintained in keratinocyte growth medium (KGM) containing supplementary growth factors (Lonza, Basel, Switzerland) at 37 °C in a water-saturated atmosphere of 5% CO₂. To examine the effect of the clinical isolates on the cell viability and proliferation of HOK-16B cells, cells (6 × 10⁴ cells) were infected with four clinical isolates of E. coli, E. coli BL21 (Thermo Fisher Scientific, Waltham, MA, USA), and S. salivarius KCTC 5512 (Korean Collection for Type Culture, Jeongeup, Jeollabuk-do, Korea) at a multiplicity of infection (MOI) of 1000 in KGM without antibiotics for 2 hours. After 2 hours, 50 μg/ml and 100 μg/ml gentamicin were added to kill extracellular E. coli and S. salivarius, respectively. After incubation for the indicated times, the cells were detached by trypsin-EDTA and enumerated after trypan blue staining. To analyze apoptosis of cells, the detached cells were also washed with Annexin-binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, pH 7.4) and stained with 5 μl of Annexin V-Alexa Fluor™ 488 conjugate (Life Technologies, Carlsbad, CA, USA) and 5 μg/ml PI in 100 μl of Annexin-binding buffer for 15 min at room temperature. After adding 400 μl of Annexin-binding buffer, the stained cells were analyzed by FACSCalibur™ (BD Biosciences, San Diego, CA, USA).

Fab proteins were expressed in E. coli BL21 (ThermoFisher), as described^{43 (link)}. Following expression, cells were harvested by centrifugation and cell pellets were flash-frozen using liquid nitrogen. The cell pellets were thawed, re-suspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1%Triton X-100, 1 mg/ml lysozyme, 2 mM MgCl₂, 10 units of benzonase), and incubated for 1 h at 4 °C. The lysates were cleared by centrifugation, applied to rProtein A-Sepharose columns (GE Healthcare), and washed with 10 column volumes of 50 mM Tris, 150 mM NaCl, and pH 7.4. Fab protein was eluted with 100 mM phosphoric acid buffer, pH 2.5 (50 mM NaH₂PO₄, 140 mM NaCl, 100 mM H₃PO₄) into a neutralizing buffer (1 M Tris, pH 8.0). The eluted Fab protein was buffer exchanged into PBS and concentrated using an Amicon-Ultra centrifugal filter unit (EMD Millipore). Fab protein was characterized for purity by SDS-PAGE gel chromatography and concentration was determined by spectrophotometry at an absorbance wavelength of 280 nm.

The GST pull-down assay was employed to detect protein-protein interactions. Briefly, the Myc-PARP1 plasmid was transfected into HEK-293T cells, followed by cell lysis with GST lysate and subsequent collection of proteins. The USP1 and USP1 C90S sequences were cloned into the pGEX-4T-1 vector to obtain GST-USP1 and GST-USP1 C90S plasmids, respectively. These plasmids were then transferred into E. Coli BL21 (DE3, EC0114, ThermoFisher Scientific) for protein expression induced by IPTG (100 μM, 34060, ThermoFisher Scientific). After collection and sonication of the E. coli cells, purified GST-USP1 or GST-USP1 C90S proteins were obtained using complete GST-Tag purification resin (08778850001, Roche). The protein was expressed and purified following the manufacturer’s instructions, utilizing BeyoGold™ GST-tag purification resin (P2251, Beyotime) for purification. The beads were washed with GST pull-down binding buffer (50 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM DTT, 10 mM MgCl2; pH 8.0), then incubated with purified GST-USP1 or GST-USP1 C90S in complex with Myc-PARP1 on a rotary windmill at 4 °C for 4 hours. Finally, the beads were washed and subjected to western blot analysis.

We used the mechanically stable talin R3-IVVI domain as a model substrate, as previously reported in force spectroscopic studies^{21 (link),22 (link)}. For purification, the protein construct was transformed into the E. coli BL21 (DE3 Invitrogen) competent cells. Cells were grown in Luria broth at 37 °C with carbenicillin, till the O.D. becomes 0.6–0.8 at 600 nm. The cultures were then induced with 1 mM Isopropyl β-d-thiogalactopyranoside (IPTG, Sigma Aldrich) overnight at 25 °C. The cells were pelleted and re-suspended in 50 mM sodium phosphate buffer pH 7.4, containing 300 mM NaCl and 10% glycerol. Phenylmethylsulfonyl (PMSF) was used as a protease inhibitor followed by lysozyme for membrane lysis. After incubating the solution for 20 min at 4 °C, the dissolved pellet was treated with Triton-X 100 (Sigma Aldrich), DNase, RNase (Invitrogen), and 10 mM MgCl₂ and kept at 4 °C in the rocking platform. The cells were disrupted in French press and cell lysate was centrifuged at 11,000 rpm for 1 h. The protein was purified from the lysate using Ni²⁺-NTA column of ӒKTA Pure (GE healthcare). For in vitro biotinylation of the polyprotein, and Avidity biotinylation kit was used and the biotinylated polypeptide was purified by Superdex-200 increase 10/300 GL gel filtration column in presence of Na-P buffer with 150 mM NaCl⁷⁸ .