Vibra cell vcx500

The process of preparing PBS films involved utilizing the solvent-casting process. Initially, 0.5 g of PBS pellets were dissolved in 250 mL of chloroform and then heated in a water bath set to 60 °C. After fully dissolving the pellets, the resultant solution was poured onto a Petri dish and allowed to stand in a fume hood at room temperature until the solvent totally evaporated.
To prepare a solid plate containing bioplastic emulsion, 0.5 g of each pellet of PBS, PBAT, PCL, PLA, PHA, PHB, and P(3HB-co-4HB) was added to 20 mL of DCM and dissolved in a water bath at 60 °C until the pellet was completely dissolved. Thereafter, 50 mL of distilled water (DW) was added to the lysate, and 1 mL of 2% Sarkosyl NL was added to the interface. The mixture was subjected to sonication using a Vibra-Cell VCX500 (Sonics & Materials, Inc., Newtown, CT, USA) with 10 s of pulsing at 30% amplitude for a duration of 10 min. Thereafter, 2% agarose and 1 g/L bioplastic emulsion were added to the marine broth (MB; Difco Laboratories, Detroit, MI, USA) medium, and this mixture was sterilized by autoclaving at 120 °C for 15 min.

Expression was performed in freshly transformed E. coli BL21 (DE3) cells grown in Terrific Broth medium and induced overnight with 0.5 mM IPTG at 20°C. Pelleted cells resuspended in phosphate buffer saline (PBS)-NaCl (PBS 1X, 1 M NaCl, pH 7.4) were lysed by sonication [80% amplitude for 150 s with 13 mm diameter probe, Vibra-Cell VCX 500 (Sonics)]. Tagged proteins were purified at 4°C by immobilized metal ion chromatography (IMAC) on a 1 ml Protino Ni-NTA column (Macherey-Nagel) using 500 mM imidazole in running buffer (50 mM Tris, 300 mM NaCl, 5% glycerol, pH 8.0) for elution, followed by size exclusion chromatography (SEC) on a Hiload 16/60 Superdex75 prep grade column (GE Healthcare Life Science) in 1X PBS. Purity of eluted proteins was assessed by Coomassie blue staining of protein separated on denaturing Tris-tricine or Tris-glycine polyacrylamide gels. Purification yields were estimated from absorbance at 280 nm based on extinction coefficients computed from protein amino acid composition. Purified proteins were adjusted to 1 mg.ml⁻¹ in 50% glycerol and stocked at −20°C.

Synthesis of lipid nanoparticles (LNPs) is done by a thin layer evaporation method [21 (link)]. In brief, DSPC, cholesterol, and polyethylenimine (PEI) (4:1:2) were dissolved in Chloroform and Methanol (1:1) and kept on magnetic stirrer for 4 hr at 25 °C and 200 rpm. Next, the solvent was evaporated using a rotary evaporator (DLAB RE-100 Pro) at 50 °C, 40 RPM, to obtain a thin layer. The obtained thin layer of lipids was dispersed in doubled distilled water by sonication (Sonics, Vibra cell VCX 500) at 50 amplitude (10 s on-off cycle) for 10 min and extruded using a 200 nm then 100 nm polycarbonate membrane at 50 °C to control the LNPs size.

For the preparation of the media plates containing bioplastics (i.e., polylactic acid; PLA, polybutylene succinate; PBS, polybutylene adipate terephthalate; PBAT, polycaprolactone; PCL, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-HV)]), 0.2 g of the bioplastic pellets was dissolved in 40 mL of DCM in a 60 °C water bath for 2 h. After the bioplastics had dissolved in DCM, 2 mL of 2% Sarkosyl NL and distilled water were added and mixed thoroughly using a Vibra-Cell VCX500 (Sonics & Materials, Inc., Newtown, CT, USA) with 15 s of pulse and an amplitude of 40% for 10 min. As a result, the dissolved bioplastics in the solvent phase were uniformly emulsified in the water phase, and the mixture became an opaque emulsion [11 (link)]. After sonication, the solvent was completely evaporated in a fume hood using a stirrer at 60 °C to prevent cell damage due to the remaining solvent. Finally, marine broth (MB) and agarose were added and autoclaved [12 (link),13 (link)].

Firstly, ZnO nanotube was prepared via microwave technique in presence of poly vinyl alcohol (PVA) as a stabilizing agent. Aqueous solution of zinc acetate (14 mM) Zn(CH₃COO)₂·2H₂O (Rankem, Gurgaon, India) was prepared by dissolving 8 g of zinc acetate in 150 mL of distilled water. Then, 25 mg of PVA (Sigma-Aldrich, Darmstadt, Germany) was mixed with zinc salt as a stabilizing agent, and sodium hydroxide (NaOH) (Sigma-Aldrich, Darmstadt, Germany) was added for zinc salt reduction. The prepared aqueous solution was maintained in a microwave (THOMSON-COMBI1, Thomson Premier Lighting & Appliance, Logan, UT, USA) for 1 h at 800 W. Subsequently, for magnetite immobilization, 0.5 g of the prepared nano-zinc oxide was suspended using a direct sonication probe ultrasonic homogenizer (Vibra-Cell VCX 500, SONICS, Newtown, CT, USA) in 200 mL mixed solution of iron (III) chloride and iron (II) sulphate with a molar ratio of 2:1 until homogeneous suspension was obtained. A sodium hydroxide solution of 5 M was added dropwise to the previous suspension at 70 °C and maintained for 30 min under constant stirring until black precipitate of magnetic zinc oxide synthesised. The obtained black powders were washed several times with distilled water and absolute ethanol, and then separated using centrifugation at a force at 4000 rpm. Finally, the nanopowders were dried at 70 °C overnight.

Boron-included DNDs were sourced from SkySpring Nanomaterials, and classified according to the data-sheet as 3–4 nm in size. The dry powder was placed in DI water at a concentration of 0.05 g/l, then ultra-sonicated on 40% power for 5 hours (Sonics Vibracell VCX 500; 500 W, 20 kHz). The solution was used to seed the DNDs on either RCA cleaned silicon substrates or H-terminated silicon substrates (microwave hydrogen plasma, Seki Technotron, AX1010, 1 kW, 20 mbar, 800 C, 20 minutes). Seeding was by immersion in an ultra-sonic bath for 3 minutes. In other cases the dry powder was used to create solid round pellets (11 mm diameter, 3 mm thick) using a mechanical press (10 T weight).