Sorvall legend micro 21r centrifuge

The above overnight grown culture was used for further inoculation in fresh LB broth. The expression of rOmp2a protein was done by induction with IPTG (isopropyl-β-D thiogalactopyranoside) using different concentrations (0.2 mM, 0.5 mM, 1 mM, 1.2 mM and 1.5 mM) and final concentration of induction was optimized with 1 mM IPTG (Sigma) at ~0.6 OD after 3 h of fresh inoculum of culture. The induced and un-induced fragments were collected at different intervals of 3 h, 5 h and overnight induction and further run on SDS-PAGE (12%) according to the protocol of Laemmli [14 (link)] (Fig. 2). To check whether the protein is soluble or forming inclusion bodies, 5 ml of induced pellet was dissolved in lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole and 1 mg/ml of lysozyme). The lysed suspension were incubated on ice and then sonicated at 40 W amplitude for 10 min and 8 s of pulse (Vibra cell, Sonics, USA). The sonicated suspension was then centrifuged at 9600 x g for 10 min at 4 °C (Sorvall Legend Micro 21R Centrifuge, Thermo Scientific). The pellet and supernatant was collected and further checked for the expressed recombinant protein by SDS-PAGE analysis.

A selection plasmid containing an inducible bacterial toxin and target site was transformed into NEB^® Turbo Competent E. coli (High Efficiency) (S7 Fig). We started an 8 mL overnight culture of the cells shaking at 250 rpm at 37°C for 16 hours. In the morning, 200 μL of cells were then inoculated into 50 mL of Terrific Broth (Teknova) with 2.5 μL of 1X10¹¹ pfu/ml M13KO7 Helper Phage (New England Biolabs). After four hours of growing the culture at 37°C at 250 rpm, cells were spun at 4500 g for 10 min in an Avanti JXN-26 centrifuge (Beckman Coulter). The supernatant was collected without disturbing the cell pellet and the centrifugation was repeated. The top 40 mL of supernatant were added to a fresh tube and we mixed with 10 mL of 2.5 M NaCl/20% PEG-8000 (w/v) (VWR). Phages were precipitated overnight at 4°C for 16 hours and then spun at 12,000 g for one hour at 4°C. Pellet was resuspended in 1 mL of TBS and 200 μL of 2.5 M NaCl/20% PEG-8000 (w/v) (VWR) was added. Phages were precipitated for 3 hours on ice and then spun them at 12,000 rpm in a Sorvall^™ Legend^™ Micro 21R centrifuge (Thermo Scientific) for 30 minutes at 4°C. After removing the supernatant, 200 μL of 1X TBS and 200 μL of 50% glycerol were used to resuspend the pellet. The phages were stored at -20°C until the start of the selection.

The amphiphilic triblock copolymer [P(MeOx₃₇-b-PBuOx₂₁-b-PMeOx₃₆)), Mn = 8.25 kg/mol, PDI (DM = 1.21)] was synthesized as reported previously by using living cationic ring-opening polymerization and used to prepare LDN-57444 loaded polymeric micelle formulation (LDN-POx) via the thin film hydration method [39 (link)]. Briefly, predetermined amounts of polymer and LDN-57444 were each dissolved in organic solvents and mixed together. The organic solvent was then evaporated under a stream of nitrogen gas (50 °C) to form the thin film. To completely remove the residual organic solvent, the films were deposited in the vacuum chamber (approx. 0.2 mbar) overnight. Subsequently, the thin films were rehydrated with saline and then incubated at room temperature for 10 min to produce LDN-57444 loaded polymeric micelle formulations. The aqueous polymeric micelle formulation was centrifuged at 10,000 rpm for 3 min (Sorvall Legend Micro 21R Centrifuge, Thermo Scientific) to precipitate non-dissolved drug or drug-polymer aggregates. The transparent supernatant solutions of micelle samples were used for the further analysis. The hydrodynamic diameter and polydispersity index (PDI) of LDN-57444 loaded polymeric micelles were determined by dynamic light scattering (DLS) using Malvern Nanosizer.