One shot cell disrupter

After amplification using primers PmrAF and PmrAR, PCR products were inserted into pET-28a(+) digested with NdeI and HindIII to add a carboxyl terminal 6-his-tag. Plasmids were transformed into the expression strain E. coli BL21 (DE3) and cultured in LB medium (5 ml) at 37°C overnight. Cells were diluted 1:100 into 1 L LB broth containing ampicillin and cultured. When OD₆₀₀ was between 0.4 and 0.5, recombinant PmrA was activated with 0.1 mM IPTG at 16°C for 20 h.
Mixtures were centrifuged at 7,000 × g for 20 min at 4°C and resuspended in lysis solution, followed by harvest of 6 L of cells and disruption by a One Shot Cell Disrupter (Constant Systems, Daventry, UK). To isolate insoluble substances, mixtures were passed through 0.45-μm syringe-end filters after centrifuging at 12,000 × g for 20 min at 4°C before loading on a His-tag column. Supernatants were transferred to a Ni-NTA affinity chromatography column (GE Healthcare, Pittsburgh, PA, USA). After balancing the column with lysis buffer, recombinant protein was added and eluted using washing buffer with an imidazole concentration gradient (0, 30, 60, 100, 200, and 500 mM). Washing buffer (30 ml) containing target recombinant proteins was dialyzed in 20 mM Tris-HCl, pH 8.0. Proteins were subjected to SDS-PAGE. Unstained and prestained protein markers (Fermentas, SM0431 and SM0671 Waltham, USA) were applied as references.

Riboflavin concentration was assayed in cell extracts and culture supernatants. 50 mL of the culture was centrifuged at 9.000 × g for 10 min at 4°C. The supernatant was filtered through a 0.22 μm filter and frozen at -20°C. The biomass was washed with the same volume of 0.1 M Na-phosphate buffer pH 7.0. The pellet was resuspended in the buffer (1/10 of the initial volume of the culture), and the suspension was frozen at -20°C. The thawed biomass was passed through the One Shot Cell Disrupter (Constant Systems, Ltd., Daventry, United Kingdom) at 40 KPsi. The cell extract, obtained by centrifugation of the sample at 13.000 × g, 15 min, 4°C was maintained at -20°C.
Riboflavin concentration was quantified using a microbiological bioassay with Lactobacillus casei subsp. rhamnosus ATCC 7469 as test organism, based on Woodson et al. (1946) (link). Growth of the test organism was measured in 5 mL of Riboflavin Assay Medium (BD, Flanklin Lakes, NJ, United States) supplemented with 5 mL of properly diluted sample and compared with a calibration curve in the range of 0–300 ng mL^–1, according to the protocol described by the medium manufacturer. Microbiological assay measurements were replicated at least six times.

E. coli-optimized EcLpxH and KpLpxH sequences, cloned into a pET-26b(+) vector (Novagene) using NdeI/XhoI restriction sites, were ordered from GenScript Biotech (the Netherlands), giving constructs with a His₆-tag at the C-terminal end, and bearing kanamycin resistance. Competent E. coli BL21-AI cells (Invitrogen) were transformed and grown at 37 °C in Luria broth containing 50 μg/mL kanamycin to an OD₆₀₀ of 0.5 to 1.0, then induced for 2 to 3 h by adding IPTG and L-arabinose to final concentrations of 0.5 mM and of 2 mg/mL, respectively. Harvested cells were lysed in a One-Shot cell disrupter (Constant Systems Ltd., UK) in 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, 4 M urea, 5% (v/v) glycerol, 0.01 mg/mL RNase A and 0.02 mg/mL DNase, plus cOmplete, EDTA-free protease inhibitor cocktail (Roche). Proteins were purified using Ni-nitrilotriacetic acid agarose (Qiagen), HiTrap Heparin HP affinity chromatography (Cytiva), and HiLoad 16/60 Superdex 200 chromatography (Amersham Biosciences). Final samples were concentrated to 15 mg/mL protein and stored in aliquots in 20 mM Tris-HCl (pH 8.0) containing 300 mM NaCl and 2 mM DTT at −80 °C. Assays were performed essentially as described by ref. 26 (link). Additional assay details are available in SI Appendix.

Cell samples from the Psip1 overexpression were resuspended in 50 mL lysis buffer (10 mM HEPES, 250 mM NaCl, 0.5 mM TCEP, 5% Glycerol, 1 tablet of cOmplete™ Protease Inhibitor Cocktail, and 1X BugBuster® Protein Extraction Reagent) and then lysed using a One-Shot Cell disrupter (Constant Systems) 2× at 20 kilopounds per square inch (kpsi). Lysed samples were collected by centrifugation at 2880×g for 10 min at 4 °C (Eppendorf Centrifuge 5810R) to separate the soluble and insoluble fractions. The soluble supernatant fraction was collected and filtered through a 0.22 µm pore-size Whatman® Puradisc filter (GE Healthcare). Filtered lysate was purified using a nickel-affinity purification column (Roche). Protein concentration was measured using the Bradford assay (65 (link)). To purify Psip1 from inclusion bodies, the protocol of Palmer and Wingfield was used (66 ). Briefly, inclusion bodies were washed three times in 100 mM Tris, pH 7.5, containing 2 M Urea, 1% v/v Triton X-100, and 5 mM EDTA, prior to being washed twice in 100 mM Tris, pH 7.5, containing 5 mM Urea. Inclusion bodies were then solubilized in 50 mM Tris, pH 7.5, containing 8 M Guanidine hydrochloride, and dialyzed overnight in 10 mM Tris, pH 7.5, containing 250 mM NaCl, 10 mM CaCl₂ and 1 µM FeCl₃.

Lactococcus lactis MG1363 ΔacmA and L. lactis UKLc10 ΔftsH TP712 lysogens were grown to OD_600nm of 0.5 and the prophage induced with 2 μg/ml of MitC. Samples were collected at 0, 30, 60, 90 (onset of lysis) min and further at 120 and 180 min in the case of the ΔftsH mutant. Cells were cooled on ice, collected by centrifugation (8000 g, 15 min, 4°C), washed with cold 50 mM sodium phosphate buffer pH 6.8 and concentrated in the same buffer at a cell density of 30 OD_600nm units per ml. Cells (2 ml) were broken twice in the One Shot Cell Disrupter (Constant Systems LTD, UK). The insoluble fraction was pelleted by centrifugation at 4°C, 8000 g for 10 min and boiled for 3 min in 1/30 of the initial volume in SDS-PAGE loading buffer. For zymograms, SDS-PAGE gels were prepared incorporating either L. lactis NZ9000 or L. lactis ΔftsH autoclaved cells to the gel matrix as previously described (Lepeuple et al., 1998 (link)). A total of 15 OD equivalents were loaded per lane. After electrophoresis, gels were washed three times with milliQ H₂O for 10 min at room temperature and incubated overnight at 37°C, with gentle shaking, in 50 mM MES-NaOH pH 6.0, and 0.1% triton X-100. Gels were briefly washed with milliQ H₂O and photographed without further staining with methylene blue.

The dried macroalgae biomasses were dispersed in distilled water at 10 g/L and vigorously mixed in a vortex (Vortex 3, IKA, Staufen, Germany) to ensure the homogeneity of the macroalgal samples. A high-pressure homogenization method using a One-Shot Cell Disrupter (Constant Systems Ltd., Warwickshire, UK) was applied to the red macroalgae suspension in one pass at a pressure of 2700 bar [35 (link)]. The working volume in this study was fixed at 8 mL. In this process, the macroalgal cells are forced to flow through a very small orifice under high-pressure conditions, and, as a result, they could be disrupted by synergistic mechanical effects, such as cavitation, turbulence, and shear stress [38 (link)]. Water-soluble protein extracts were obtained by centrifugation (6000× g, 20 min, 4 °C).

The BnUBC13 open-reading frames (ORFs) were isolated from pGBT-BnUbc13s and cloned into pET30a (Novogene) as a His₆ fusion. The resulting pET-BnUbc13s were co-transformed with pGEX-AtUev1D [22 (link)] into Epicurian coli BL21-CodonPLus (DE3)-RIL strain (Thermo Sci. 2,287,225). The transformed cells were cultured overnight in LB + Amp + Kan, diluted 1:50 into fresh culture and incubated until OD_{600 nm} of 0.6–0.8. The His₆-BnUbc13s and GST-AtUev1D fusion proteins were induced by adding isopropyl-b-D-thiogalactopyranoside (IPTG) to the final concentration of 0.2 mM and the incubation continued for 6 h. The cells were harvested by 8,000 rpm centrifugation in a Beckman Coulter Avanti JA17 rotor for 1 h at 4 °C, resuspended in phosphate-buffered saline (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.3), and passed through Constant Systems one shot cell disrupter at 25 PSI. The resulting crude extract was centrifuged at 17,000 rpm in the same JA17 rotor for 30 min at 4 ^oC, and the soluble fraction was used for the GST pulldown assay.