Details of the designs of adapted RCs are described in Supplementary Note 1 . All adapted RCs were expressed in a strain of Rba. sphaeroides engineered to lack light-harvesting complexes54 (link),55 (link). Bacterial cells grown under dark/semiaerobic conditions and harvested by centrifugation were suspended in 20 mM tris(hydroxymethyl)aminomethane (Tris, pH 8.0), supplemented with protease inhibitor and DNAase, and lysed in a cell disruptor (Constant Systems) at 20,000 psi. Cell debris was removed by centrifugation at 18,000 rpm for 15 min at 4 °C. The supernatant was then incubated in the dark at 4 °C for 1 h with 1.5% (v/v) lauryldimethylamine N-oxide (LDAO) and 200 mM NaCl. After ultracentrifugation at 38,000 rpm for 30 min at 4 °C the supernatant was collected and initial purification carried out using a Ni-NTA (nitrilotriacetic acid) column (GE Healthcare). The protein eluate was concentrated and then further purified using a Superdex 200 gel filtration column (GE Healthcare) in 20 mM Tris (pH 8) containing 0.04% N-dodecyl-β-d -maltopyranoside (Tris/DDM buffer). Eluted fractions with a low 280 to 804 nm absorbance ratio (<1.5) were pooled, concentrated and stored at −80 °C.
Cell disruptor
Manufactured by Constant Systems
Sourced in United Kingdom
A cell disruptor is a laboratory equipment used to physically break down the cell walls or membranes of biological samples, such as cells, tissues, or microorganisms. It facilitates the extraction and isolation of intracellular components like proteins, nucleic acids, or organelles for further analysis or experimentation.
Automatically generated - may contain errors
Lab products found in correlation
Hitrap, by Cytiva (4 mentions)
Nickel resin, by Qiagen (3 mentions)
Histrap hp column, by GE Healthcare (3 mentions)
Ni nta agarose, by Qiagen (3 mentions)
Histrap hp column, by Cytiva (3 mentions)
Superdex 200, by GE Healthcare (2 mentions)
Superdex 200, by Qiagen (2 mentions)
Kta system, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Constant Systems (2 mentions)
Dnase 1, by Promega (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Deae cellulose column, by Merck Group (2 mentions)
Hiload 26 60 superdex 75 column, by GE Healthcare (2 mentions)
Sorvall rc 6, by Thermo Fisher Scientific (2 mentions)
Superdex 200 pg 16 60 size exclusion column, by Cytiva (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
α flag m2, by Thermo Fisher Scientific (2 mentions)
Ti45 rotor, by Beckman Coulter (2 mentions)
Ni nta, by GE Healthcare (1 mentions)
Specord 250 plus, by Analytik Jena (1 mentions)
86 protocols using cell disruptor
1
Purification of Adapted Reaction Centers
2
Purification of IsiE-3xFLAG Protein from E. coli
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Escherichiacoli BL21 (DE3) carrying the plasmid pOEIsiE-FLAG was cultured in LB medium at 37°C, 180 r.p.m. to an OD600 of 0.8, and then induced with 0.5 mM IPTG for 4 h. Cells were collected by centrifugation at 3,000x g for 5 min, and washed with lysis buffer (137 mM NaCl, 12 mM phosphate, 2.7 mM KCl, 1X cOmplete™ protease Inhibitor, pH 7.4). Washed cells were spun again and suspended in 1/10 of the initial culture volume in lysis buffer. Cells were physically disrupted by high pressure using a Constant Systems Cell Disruptor. Cell lysate was centrifuged at 4°C, 16,000 x g for 30 min to remove unbroken cell and membrane fractions. The supernatant was filtered through a 0.22-μm-pore-size filter and used to purify IsiE-3xFLAG by specific binding to ANTI-FLAG® M2 magnetic beads according to the manufacturer’s instructions. Absorption spectra were measured at room temperature using Specord® 250 Plus (Analytik Jena) spectrophotometer. The purified IsiE-3xFLAG protein was dissolved in PBS buffer (137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl, pH 7.4), while a solution of 3X FLAG peptide using the same buffer was used as blank control.
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Escherichiacoli BL21 (DE3) carrying the plasmid pOEIsiE-FLAG was cultured in LB medium at 37°C, 180 r.p.m. to an OD600 of 0.8, and then induced with 0.5 mM IPTG for 4 h. Cells were collected by centrifugation at 3,000x g for 5 min, and washed with lysis buffer (137 mM NaCl, 12 mM phosphate, 2.7 mM KCl, 1X cOmplete™ protease Inhibitor, pH 7.4). Washed cells were spun again and suspended in 1/10 of the initial culture volume in lysis buffer. Cells were physically disrupted by high pressure using a Constant Systems Cell Disruptor. Cell lysate was centrifuged at 4°C, 16,000 x g for 30 min to remove unbroken cell and membrane fractions. The supernatant was filtered through a 0.22-μm-pore-size filter and used to purify IsiE-3xFLAG by specific binding to ANTI-FLAG® M2 magnetic beads according to the manufacturer’s instructions. Absorption spectra were measured at room temperature using Specord® 250 Plus (Analytik Jena) spectrophotometer. The purified IsiE-3xFLAG protein was dissolved in PBS buffer (137 mM NaCl, 10 mM Na2HPO4, 2 mM KH2PO4, 2.7 mM KCl, pH 7.4), while a solution of 3X FLAG peptide using the same buffer was used as blank control.
3
Purification of Bacterial Cytotoxins
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Purification of typhoid toxin and cytolethal distending toxin (CDT) was conducted as described previously [1 (link), 72 (link)]. Briefly, the genes encoding typhoid toxin in Salmonella Typhi (pltA/pltB/6xHis-cdtB) or CDT in Campylobacter jejuni (cdtA/cdtC/6xHis-cdtB) were cloned into the pET28a (Novagen) expression vector. Escherichia coli strains carrying the different plasmids were grown at 37°C in LB media to an OD600 of ~0.6, toxin expression was induced by the addition of 0.5 mM IPTG, and cultures were further incubated at 25°C overnight. Bacterial cell pellets were resuspended in a buffer containing 15 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1 mg/ml DNase, 0.1 mg/ml lysozyme, and 0.1% PMSF and lysed by passage through a cell disruptor (Constant Systems Ltd.). Toxins were then purified from bacterial cell lysates through affinity chromatography on a Nickel-resin (Qiagen), ion exchange, and gel filtration (Superdex 200) chromatography as previously described [1 (link), 72 (link)]. Purified toxins were examined for purity on SDS-PAGE gels stained with coomassie blue.
4
Isolation and Purification of Bacterial Outer Membrane
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Bacterial cultures (50 mL), grown in the presence or absence of stress, were incubated according to the optimum micC/ompN induction conditions determined by the β-galactosidase assay. The cells were washed and concentrated 12.5 fold in 20 mM sodium phosphate buffer (pH 7.4), and lysed by one passage through a cell disruptor (Constant Systems) at 2 kbar. After removal of cell debris by centrifugation (7000× g, 20 min, 4 °C) the supernatant was ultracentrifuged (100,000× g, 60 min, 4 °C) to collect the whole cell envelopes. These were resuspended in 0.3% N-laurylsarcosinate, and incubated for 30 min at room temperature to solubilize the IM. The insoluble OM extracts were pelleted by centrifugation (100,000× g, 60 min, 4 °C).
5
Purification of Recombinant HsRecA Proteins
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All purification steps were carried out at 4°C. The frozen cells were thawed on ice and lysed in a cell disruptor (Constant Systems Ltd., UK) in 25–50 mL buffer ANI (25 mM NaH2PO4 pH 7.0, 5% glycerol, and 0.5 M NaCl) containing a tablet of a cocktail of protease inhibitors. The soluble protein extract was loaded onto a 5 mL Hi-Trap Chelating column (GE Healthcare) charged as indicated by the manufacturer. After being submitted to an imidazole gradient (0.04–0.8 M) in buffer ANI, the fractions containing the HsRecA or the HsRecA L53Q proteins were dialyzed and stored at -80°C after freezing in liquid nitrogen.
6
Inulinase Activity Assay Protocol
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Inulinase activity was assayed by determining the concentration of released reducing sugars according to Hu et al. [10 (link)] with minor modification. One unit of inulinase activity was defined as the amount of enzyme that produces 1 μmol fructose per min under the assay conditions. The reaction mixture containing 50 μL of culture supernatant and 450 μL of 2 % inulin (dissolved in 0.1 M acetate buffer, pH 5.0) was incubated at 50 °C for 15 min. The reaction was terminated at 100 °C for 10 min, and the concentration of reducing sugar in the mixture was determined [45 (link)]. Absorbance was read at a wavelength of 540 nm. To detect intracellular inulinase activity, yeast cells harvested from 1 mL culture were washed and resuspended into 2 mL sodium acetate buffer (pH 5.4) supplemented with 1/100 (v/v) protease inhibitor cocktail (Ameresco). Afterward, the yeast cells were disrupted at 35 kpsi by three shots using a cell disruptor (Constant Systems Ltd.). Next, the disrupted cell suspension was centrifuged at 10,000g for 5 min, and the supernatant was used for intracellular inulinase activity assay.
7
Recombinant Protein Purification Protocol
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For the overexpression His-tagged cyAbrB1 and cyAbrB2, 10 milliliters of E. coli BL21(DE3) overnight cultures containing pNHis-0822 or pNHis-0359 were inoculated into 1 L of LB medium supplemented with ampicillin and cultured at 37°C with continuous agitation. The expression of recombinant proteins was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG) to a concentration of 0.5 mM when the OD600 reached 0.6. After 4 h, the cells were collected by centrifugation at 3,000 g for 10 min. Pellets were resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.5) supplemented with Protease Inhibitor Cocktail and then broken using a cell disruptor (Constant Systems Limited). The lysates were centrifuged at 12,000 g for 30 min, and the supernatants filtered through a 0.45 μm filter. The recombinant proteins were then purified using a 1 mL HisTrap HP column and ÄKTA system (GE Healthcare) according to the manufacturer’s instructions. For the overexpression of His-tagged RpaB, a similar procedure was carried out but using E. coli BL21(DE3) pLysS as described previously (31 (link), 46 (link)).
8
Protein Purification for MitoLuc Import Assays
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Proteins were purified for use in MitoLuc import assays, as detailed previously (Needs et al., 2023 (link)). Protein expression was carried out as described previously (Pereira et al., 2019 (link)). A single colony of transformed BL21 (DE3) bacteria was grown in LB medium with appropriate antibiotic overnight (37°C; 200 rpm). Pre-cultures were used to inoculate a secondary culture at a 1:100 dilution, in 2× yeast extract tryptone (YT) supplemented with appropriate antibiotics. Secondary cultures were grown until mid-log phase, then induced with 1 mM IPTG or 0.2% (w/v) arabinose and grown for a further 3 h. Cells were harvested by centrifugation (15 min; 6000 g ), resuspended in TK buffer (20 mM TRIS base, 50 mM KCl; pH 8.0), cracked in a cell disruptor (Constant Systems; 2 cycles at 25 kpsi), and clarified by centrifugation (45 min; 103,500 g ).
9
Purification of His-tagged CspC Proteins
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To purify proteins, E. coli BL21 (DE3) strains harboring plasmids expressing His6-tagged wild-type or variant CspC proteins were grown in LB at 37°C for 3 h and 0.7 mM of IPTG was added to induce gene expression and further incubated at 30°C for 3 h. Cells were collected and washed twice with a solution containing 50 mM Tris–HCl (pH 8.0) and 150 mM NaCl. Washed cells were resuspended in solution A [50 mM Tris–HCl (pH 8.0), 150 mM NaCl] containing 150 μg ml–1 lysozyme, 1 mM MgCl2, DNase I (Promega) and EDTA-free protease inhibitor cocktail (Roche), and incubated at 4°C for 30 min. Cells were broken using Cell Disruptor (Constant Systems Ltd). After adding imidazole to 20 mM as final concentration, cell debris was removed by centrifugation (12 000×g, 30 min) and the supernatant was applied to Ni-Nta agarose (Qiagen) column. The column was washed with solution A containing 25 mM imidazole, and proteins were eluted with solution A containing 100–300 mM imidazole and dialyzed with the same solution without imidazole.
10
Isolation of P. chromatophora Chromatophores
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P. chromatophora CCAC0185 [axenic version (7) ] was grown (11) and chromatophores isolated as described previously (10) . In brief, P. chromatophora cells were washed three times with isolation buffer (IB: 50 mM HEPES pH 7.5, 2 mM EGTA, 2 mM MgCl2, 250 mM sucrose, 125 mM NaCl) and depleted of dead cells on a discontinuous 20-80% Percoll gradient. The resulting pellet of intact cells was resuspended in IB, cells were broken in a cell disruptor (Constant Systems) at 0.5 kbar, and intact chromatophores isolated on another discontinuous 20-80%
Percoll gradient. To increase purity, isolated chromatophores were re-isolated from a fresh Percoll gradient. Recovered chromatophores were washed three times in IB, supplemented with protease inhibitor cocktail (Roche cOmplete), frozen in liquid nitrogen, and stored at -80°C until further use.
Percoll gradient. To increase purity, isolated chromatophores were re-isolated from a fresh Percoll gradient. Recovered chromatophores were washed three times in IB, supplemented with protease inhibitor cocktail (Roche cOmplete), frozen in liquid nitrogen, and stored at -80°C until further use.
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