Vivaspin turbo 15

To understand the mechanism(s) of T4 phage inactivation by EcN, E. coli strains were subjected to different treatments as mentioned below. (i) Heat-killing: 10 ml of 24 h static cultures were pelleted at 4696 × g for 10 min at RT and washed twice with 500 μl of 0.9% saline. The pellets were resuspended in LB medium to obtain 10¹⁰ Cfus/ml and thereon heat-killed for 1 h at 100°C. The supernatants of heat-killed cells were sterile filtered (0.22 μm PALL filter, Cat no: 514-4131, VWR). (ii) Concentrated supernatant: supernatants of 24 h E. coli static cultures were sterile filtered and concentrated approximately 10 times (10×) with Vivaspin Turbo 15 (MWCO: 5 KDa, Cat no: VS15T11, Sartorius). (iii) LPS destruction: Further to understand the involvement of LPS, E. coli heat-killed cultures (10¹⁰ Cfus/ml) or supernatants were incubated with 25 μg/ml polymyxin B (PMB) for 1 h at 37°C. 100 μl of the differentially treated E. coli strains or supernatants were added to 100 μl of phages, to a final volume of 1 ml with LB medium and incubated for 24 h at 37°C in a 24 well system. In the case of PMB treatment, 25 μg/ml PMB was used also in the coincubation.

The preparation of semi-pure cellobiose dehydrogenase was carried out using a two-step purification strategy according to Sulej et al. (2019) [51 (link)]. Briefly, the crude enzyme was precipitated with ammonium sulfate fractionation in the saturation ranges of 40–90% (P. sanguineus), 30–50% (P. lindtneri), 15–85% (C. unicolor), and 20–80% (P. chrysosporium) at 0 °C. The precipitates were dissolved in deionized water and by diafiltration through centrifugal concentrators (Vivaspin Turbo 15) in polyethersulphone (PES) with a cut-off of 30 kDa (Sartorius, Göttingen, Germany). The diafiltrated sample was loaded onto a DEAE Sepharose (fast flow) column for anion exchange chromatography. Fractions with high CDH activity were pooled, desalted, and used as a partially purified enzyme for the immobilization experiments. The protein concentration during enzyme purification was monitored by ultraviolet (UV) absorbance at 280 nm.

The coding region of GCaMP6m was moved into a constitutive bacterial expression vector (pCP, generous gift from Nathan Shaner), using ligation-independent cloning [49 (link)]. E. coli colonies expressing GCaMP6m were picked for presence of 470 nm excited fluorescence and grown at 34°C for 48 hours in Circlegrow (MP Biomedicals) with Ampicillin. Bacterial pellets were lysed using BugBuster (Novagen) and Benzonase (Novagen). Cleared lysates were then His-tag purified using Protino Ni-TED 2000 packed columns (Macherey-Nagel). Purified fluorescent proteins were eluted in 1x PBS with imidazole pH 8 buffer solution. The protein was then concentrated and buffer exchanged into a pH 7.2 MOPS buffer using Vivaspin Turbo 15 (Sartorius) polyethersulfone ultrafiltration columns. The same buffer exchange columns were used to make all Ca²⁺-free and Ca²⁺-saturated samples for protein measurements, except for the Ca²⁺ titration experiments. All purified GCaMP6m Ca²⁺-free protein measurements for this work were carried out in 10 mM EGTA /100 mM KCl / 30 mM MOPS at pH 7.2. All purified GCaMP6m Ca²⁺-saturated protein measurements for this work were carried out in 10 mM CaEGTA /100 mM KCl /30 mM MOPS at pH 7.2.

Bone samples are decalcified in 0.5M HCl at 4 °C for several hours (powder) until no CO₂ effervescence is observed or for several days/weeks (whole bone) until CO₂ effervescence has stopped and the sample is soft/translucent (HCl changed once or twice a week). 0.1M NaOH is added for 30 min to remove humic acids, followed by 0.5M HCl for 15 min. The resulting solid is gelatinised in HCl pH 3 in a heater block at 75 °C for 20 h. The gelatin is then filtered with an Eeze-Filter™ (Elkay Laboratory Products (UK) Ltd.) to remove small (>80 µm) particles (cleaned as above). The gelatin is then ultrafiltered (Sartorius Vivaspin Turbo 15 with a Polyethersulfone Membrane and 30 kDa MWCO). Prior to use, the filters are centrifuged twice for 10 min with Milli-Q water, followed by 1 h submerged in Milli-Q water in an ultrasonic bath, followed by three time 10 min rinses with Milli-Q water in the centrifuge (Bronk Ramsey et al. 2004 ; Brock, Bronk Ramsey, and Higham 2007 ). The >30 kDa and <30 kDa fractions were frozen for 24 h and then lyophilised for 48 h, and weighed immediately on a microbalance to determine the % yield.

We previously found that cl-Par-4 at pH 7.0 requires a high salt concentration to form compact helical tetramers [38 (link),39 (link)]. For NMR analysis, high salt and tetramer formation are problematic in terms of the signal-to-noise ratio and line width. Therefore, for NMR analysis, we employed the conditions of pH 4, which we have shown can induce a compact helical dimer at low salt concentration [37 (link)]. To change sample conditions to pH 4.0 with low salt while avoiding precipitation near the protein pI of 5.39, a step-wise dilution/titration procedure was used: 1.0 mL of concentrated (>10 mg/mL) cl-Par-4 in pH 7.0 buffer (10 mM Tris, 1 M NaCl, 1 mM TCEP, pH 7.0) was added dropwise to a solution of 49.0 mL of pH 3.9 buffer (10 mM Tris, 10 mM NaCl, 1 mM TCEP, pH 3.9) with constant stirring. The result was a 50 mL solution of diluted cl-Par-4 (~0.2 mg/mL) at pH 4.0 with 30 mM NaCl. This was then filtered using a 0.22 µm steriflip (Millipore Steriflip, Millipore-Sigma, Darmstadt, Germany), followed by concentration of the protein to 0.5 mM (12 mg/mL) using a 10 kDa MWCO Vivaspin Turbo 15 centrifugal protein concentrator (Sartorius, Epsom, UK). To prevent precipitation upon the addition of D₂O to the pH 4.0 cl-Par-4 NMR samples, D₂O was titrated to pH 4 in NMR buffer before being added to the NMR samples for deuterium lock purposes.

Expression and purification of MrNVLP were performed as described by Goh et al.^{18 (link)}. The carboxylate group of FA was activated by incubating FA (4 mM) with EDC (2.4 mM) and sulfo-NHS (2.4 mM) in HEPES buffer (25 mM HEPES, 150 mM NaCl, pH 7.4) for 2 h at 25 °C. The mixture (1 mL) was then incubated with the purified MrNVLP (1 mL, 3 mg/mL in HEPES buffer, pH 7.4) for 2 h at 4 °C. Next, the mixture was dialysed in HEPES buffer (1 L, two times) at 4 °C to remove excess FA, EDC, and sulfo-NHS. The dialysed mixture (1 mL) was layered on top of a sucrose density gradient [8–50% (w/v)] and centrifuged at 150,000 × g for 4.5 h at 4 °C. The sucrose gradient was fractionated and the concentration of FA-MrNVLP in each fraction was determined using the Bradford assay^{52 (link)}. The fractions that contained FA-MrNVLP were pooled, dialysed in HEPES buffer at 4 °C, and concentrated with the Vivaspin Turbo 15 (10 kDa cut off, polyethersulfone membrane; Sartorius, Germany). UV-visible measurement of MrNVLP and FA-MrNVLP was performed using a spectrophotometer (Jenway 7315, Cole-Parmer, U.K.). A₃₆₀ and an extinction coefficient of 5312 cm⁻¹ M⁻¹ were used to calculate the FA conjugation efficiency (CE) using equation (1). The number of FA per MrNVLP (N_FA) was calculated using equation (2). $$\mathrm{CE}\%={\mathrm{Weight}}_{\mathrm{FA}}/{\mathrm{Weight}}_{\mathrm{MrNVLP}}\times 100\%$$ $${\mathrm{N}}_{\mathrm{FA}}=\mathrm{CE}\times \left({\mathrm{MW}}_{\mathrm{MrNVLP}}/{\mathrm{MW}}_{\mathrm{FA}}\right)$$
MW; molecular weight.