Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies

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Protocol Engineering and Production of the Light-Driven Proton Pump Bacteriorhodopsin in 2D Crystals for Basic Research and Applied Technologies

Mirko Stauﬀer 1 , Stephan Hirschi 1 , Zöhre Ucurum 1, Daniel Harder 1 , Ramona Schlesinger 2,* and Dimitrios Fotiadis 1,* 1 Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, 3012 Bern, Switzerland; mirko.stauﬀ[email protected] (M.S.); [email protected] (S.H.); [email protected] (Z.U.); [email protected] (D.H.) 2 Department of Physics, Genetic Biophysics, Freie Universität Berlin, 14195 Berlin, Germany * Correspondence: [email protected] (R.S.); [email protected] (D.F.)

Received: 6 July 2020; Accepted: 19 July 2020; Published: 22 July 2020

Abstract: The light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum is a retinal-binding protein, which forms highly ordered and thermally stable 2D crystals in native membranes (termed purple membranes). BR and purple membranes (PMs) have been and are still being intensively studied by numerous researchers from different scientific disciplines. Furthermore, PMs are being successfully used in new, emerging technologies such as bioelectronics and bionanotechnology. Most published studies used the wild-type form of BR, because of the intrinsic difficulty to produce genetically modified versions in purple membranes homologously. However, modification and engineering is crucial for studies in basic research and, in particular, to tailor BR for specific applications in applied sciences. We present an extensive and detailed protocol ranging from the genetic modification and cultivation of H. salinarum to the isolation, and biochemical, biophysical and functional characterization of BR and purple membranes. Pitfalls and problems of the homologous expression of BR versions in H. salinarum are discussed and possible solutions presented. The protocol is intended to facilitate the access to genetically modified BR versions for researchers of different scientific disciplines, thus increasing the application of this versatile biomaterial.

Keywords: bacteriorhodopsin; bioelectronics; biomaterial; bionanotechnology; Halobacterium salinarum; light-driven proton pump; protein engineering; purple membranes

1. Introduction Since its discovery in 1971 [1], the light-driven proton pump bacteriorhodopsin (BR) from the extreme halophilic archaeon Halobacterium salinarum (previously known as Halobacterium halobium) has become an intensively studied model membrane protein for proton translocation across biological membranes [2–5], photochemical events in proteins [6–9], photophosphorylation [10–12] and structural studies [13–16]. The use of BR to study proton transport across membranes in diﬀerent time scales using time-resolved serial femtosecond crystallography at X-ray free electron lasers [17–20] and as an alignment tool for NMR studies [21] highlights its signiﬁcant contribution to emerging technologies. BR natively occurs in patches in the membrane of H. salinarum, called purple membranes (PMs). These patches contain BR and lipids in a hexagonal crystal lattice, with BR forming trimers. This quaternary structure and higher order is responsible for the high thermal stability of PMs [22,23]. Interesting chemical and physical properties, i.e., photochromism, photoelectrism and

Methods Protoc. 2020, 3, 51; doi:10.3390/mps3030051 www.mdpi.com/journal/mps Methods Protoc. 2020, 3, 51 2 of 22 thermal stability make BR/PMs one of the most promising biomaterials for versatile technical and scientific applications [24]. Upon illumination with light at 568 nm, the covalently bound retinal chromophore of BR undergoes isomerization by absorbing a photon. This induces a photocycle, involving a series of conformational changes and protonation states through different, consecutive states. These spectrally distinguishable intermediates are called K, L, M and O, the last of which thermally decays back to the resting state (bR) [8]. However, if the O intermediate absorbs a second photon (λmax = 640 nm), an alternative intermediate, the thermally stable Q-state, can form enabling long-term storage of information. This feature is exploited in the application of BR in holographic associative processors [25] and volumetric optical memories [26,27]. During a complete photocycle, a proton is transported from the intra- to the extracellular side of the protein, establishing an electric current (photocurrent). This photoelectric effect is applied in photovoltaic devices [28], optical switches [29], optical logic gates [30], light sensors [31], single electron [31]- and field effect transistors [32], chemical sensors [33,34], optical microcavities [35] and may even lead to protein-based retinal implants [36]. The wild-type form of BR or purple membranes is not optimal for numerous applications; thus, mutated forms were engineered to meet specific requirements. Furthermore, variants were needed to conduct studies on mechanistic and structural aspects of BR. For example, in BR-D96N, the duration of the photocycle increases from ~10 ms to ~10 s, which allowed for studying the conformational changes by high-speed atomic force microscopy [37]. The introduction of common protein tags such as the His-tag [38,39] or the creation of fusion proteins such as BR-GFP [40] enable the use of specific biophysical and bionanotechnological techniques. By the modulation of the energetic state of certain photocycle species, BR can be tailored to specific applications. The previously mentioned BR-D96N shows a prolonged lifetime of the M state, leading to an enhanced light sensitivity, which finds application in optical and holographic recordings [28]. In contrast, BR-D85N [41] and BR-V49A/I119T/T121S/A126T [24] show the prolonged lifetime of the O-state, leading to a more efficient formation of the Q-state, which is needed for long-term information storage. BR can also be optimized for photovoltaic applications by enhancing the dipole moment of the purple membranes [24]. If BR is used in photodetector or photovoltaic devices, a high photocurrent is required for efficient operation. A possibility to enhance the photocurrent is increasing the molecular surface density of BR on the used electrode. This was accomplished by engineering the BR-M163C version, which is able to bind to the gold electrode surface via the introduced cysteine residue [42]. The genetic modification of H. salinarum was first established using a polyethyleneglycol (PEG)-mediated transformation method [43,44]. The introduction of resistance genes for the antibiotics mevinolin [45–47] and novobiocin [48,49] into haloarchaeal plasmids for the selection of transformants and the development of shuttle-vectors [50–52], which enabled mutagenesis and vector amplification in Escherichia coli, further simplified the transformation of H. salinarum. The transformation of purple membrane-deficient strains (e.g., H. salinarum L33 [53,54]), in which the bacterioopsin (bop) gene is disrupted by transposable elements [54], with plasmids containing wild-type or mutant versions of the bop gene, allowed for the homologous expression of purple membranes containing wild-type or mutant versions of BR [50,51]. In parallel, methods for heterologous expression of BR in E. coli [55–58] were developed. However, this approach requires supplementation of retinal to culture media, host-specific signal sequences for membrane targeting, which have to be proteolytically removed later, as well as the purification of BR using detergents followed by reconstitution into lipids to yield functional BR. Furthermore, it was demonstrated that functional BR can be expressed in the yeast Schizosaccharomyces pombe, without the need for added signal sequences [59,60]. Importantly, it is not possible to produce BR as purple membranes using these expression systems [55]. It is therefore not recommended to use heterologous expression for applications, which require BR organized in a 2D lattice as found only in purple membranes from halobacteria. Methods for the cultivation of H. salinarum [61,62] and the isolation of purple membranes [63] were established early in BR research and were adapted in many scientific publications, including this protocol. Methods Protoc. 2020, 3, 51 3 of 22

Here, we present a detailed and straightforward description for the production of specifically engineered PMs, from the genetic modification and cultivation of H. salinarum to the isolation of pure PMs. Furthermore, we show functional and biophysical techniques for the analysis of those PMs. These methods allow for the production of a wide range of PMs with different forms of mutations, including the addition of different detection and purification tags, the introduction of functional point mutations and the production of fusion proteins. Even chimeras of BR and other proteins were shown to be functionally active, while maintaining the structural integrity of the two-dimensional crystalline PM lattice [64]. Intrinsic problems of the H. salinarum expression system are discussed and possible solutions presented.

2. Experimental Design The time needed to perform the experiments in this protocol can strongly vary, depending on the number of iteration cycles needed to achieve a satisfactory clone. 3.1 Site-directed mutagenesis of bop in pUC18 (0.5 Day) 3.2 Insertion of bop-C-His10 into pHS blue (0.5 Day) 3.3 Ampliﬁcation of pHS blue-bop-C-His10 in E. coli DH5α (1 Week) 3.4 Preparation of H. salinarum L33 cells for transformation (2–3 Days) 3.5 Preparation of plasmid for transformation (10 Min) 3.6 Transformation of H. salinarum L33 (2 H) 3.7 Outgrowth of transformed cells (2 Days) 3.8 Plating and colony formation (1–2 Weeks) 3.9 Colony picking and cultivation (1 Week) 3.10 Glycerol stock preparation (30 Min) 3.11 Clone screening by small-volume purple membrane preparation (0.5 Day) 3.12 Evaluation of BR expression by Coomassie Brilliant blue-stained SDS-PAGE and Western blot analysis (0.5 Day) 3.13 Iterative clone-picking to optimize expression of His10-tagged bacteriorhodopsin (2–3 Weeks per iteration) 3.14 Large-volume cultivation of a selected transformed H. salinarum L33 clone (2 Weeks) 3.15 Purple membrane preparation (1.5 Days) 3.16 Detergent-mediated reconstitution of BR into preformed liposomes (2 Days) 3.17 Functional characterization of BR proteoliposomes by photoactivity assay (1 Day) 3.18 Structural characterization of purple membranes by negative stain transmission electron microscopy (1 Day)

2.1. Materials Phusion High-Fidelity PCR Kit (Thermo Fisher, Reinach, Switzerland; cat. no. F553L) • 5 -pUC-BamHI-BR (forward primer): AAA AGG ATC CGA CGT GAA GAT GGG GC (Microsynth • 0 AG, Balgach, Switzerland; custom synthesis) 3 -BR-C-His -HindIII (reverse primer): AAA AAA GCT TGA TTC AGT GGT GAT GAT GGT • 0 10 GAT GAT GGT GGT GAT GTC CGT CGC TGG TCG CGG CCG CGC (Microsynth AG, Balgach, Switzerland; custom synthesis) Primer for D96N mutation in BR-C-His10: CCG CTG TTG TTG TTA AAC CTC GCG TTG CTC • GTT G (Microsynth AG, Balgach, Switzerland; custom synthesis) The plasmid pUC18-bop, i.e., the promotor and the gene of BR (bop) inserted into the commercially • available pUC18 vector (New England BioLabs, Allschwil, Switzerland; cat. no. N3041) Gel extraction/PCR puriﬁcation kit (NucleoSpin Gel and PCR Clean-up Kit, Macherey-Nagel, • Oensingen, Switzerland; cat. no. 740609) DNA Ligation Kit (Thermo Fisher, Reinach, Switzerland; cat. no. K1423) • Methods Protoc. 2020, 3, 51 4 of 22

E.Z.N.A. Plasmid Mini Kit I (VWR, Dietikon, Switzerland; cat. no. D6943-02) • BamHI-HF (New England BioLabs, Allschwil, Switzerland; cat. no. R3136S) • HindIII-HF (New England BioLabs, Allschwil, Switzerland; cat. no. R3104S) • MethodsCutSmart Protoc. 2020 bu, 3, ffx erFOR 10X PEER (New REVIEW England BioLabs, Allschwil, Switzerland; cat. no. B7204S) 4 of 21 • DH5α competent cells (Thermo Fisher, Reinach, Switzerland; cat. no. 18265017) •• BamHI-HF (New England BioLabs, Allschwil, Switzerland; cat. no. R3136S) plasmid pHS blue (shuttle vector: E. coli and H. salinarum)[39]. See Supplementary Information •• HindIII-HF (New England BioLabs, Allschwil, Switzerland; cat. no. R3104S) for nucleotide sequence (Material S1) and vector map (Figure S1) • CutSmart buffer 10X (New England BioLabs, Allschwil, Switzerland; cat. no. B7204S) • Agarose (Sigma-Aldrich, Buchs, Switzerland; cat. no. A9539) • DH5α competent cells (Thermo Fisher, Reinach, Switzerland; cat. no. 18265017) • Trisplasmid (AppliChem, pHS blue (shuttle Bioswisstec, vector: SchaE. coliff hausen,and H. salinarum Switzerland;) [39]. See cat. Supplementary no. A1430) Information • Aceticfor nucleotide acid (Sigma-Aldrich, sequence (Material Buchs, S1) and Switzerland; vector mapcat. (Figure no. S1) 695092) •• EthylenediaminetetraaceticAgarose (Sigma-Aldrich, Buchs, acid Switzerland; (EDTA; Sigma-Aldrich, cat. no. A9539) Buchs, Switzerland; cat. no. EDS) •• Tris (AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. A1430) Luria-Bertani (LB) broth (VWR, Dietikon, Switzerland; cat. no. J106) •• Acetic acid (Sigma-Aldrich, Buchs, Switzerland; cat. no. 695092) • Kanamycin sulfate (AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. 1493) • Ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich, Buchs, Switzerland; cat. no. EDS) • BactoLuria-Bertani™ Agar (LB) (BD, broth Allschwil, (VWR, Switzerland;Dietikon, Switzerland; cat. no. 214010)cat. no. J106) • • E.Z.N.A.Kanamycin Plasmid sulfate (AppliChem, Midi Kit (VWR, Bioswisstec, Dietikon, Schaffhausen, Switzerland; Switzerland; cat. no. D6904-03) cat. no. 1493) •• ™ SodiumBacto Agar chloride (BD, Allschwil, Ph.Eur/USP Switzerland; (NaCl; cat. Schweizer no. 214010) Salinen, Pratteln, Switzerland; www.salz.ch, •• E.Z.N.A. Plasmid Midi Kit (VWR, Dietikon, Switzerland; cat. no. D6904-03) cat. no. 7731) • Sodium chloride Ph.Eur/USP (NaCl; Schweizer Salinen, Pratteln, Switzerland; www.salz.ch, cat. Magnesium sulfate (MgSO ; Sigma-Aldrich, Buchs, Switzerland; cat. no. M2643) • no. 7731) 4 • TrisodiumMagnesium citrate sulfate dihydrate(MgSO4; Sigma-Aldrich, (Merck, Zug, Buchs, Switzerland; Switzerland; cat. cat. no. no. 106448) M2643) • • PotassiumTrisodium citrate chloride dihydrate (KCl; Sigma-Aldrich,(Merck, Zug, Switzerland; Buchs, Switzerland; cat. no. 106448) cat. no. 60310) •• BacteriologicalPotassium chloride peptone (KCl; Sigma-Aldrich, (OXOID, Pratteln, Buchs, Switzerland; Switzerland; cat.cat. no. 60310) LP0037) •• Bacteriological peptone (OXOID, Pratteln, Switzerland; cat. no. LP0037) CRITICAL OtherOther peptone peptone products products might might not work not work optimally. optimally. • SodiumSodium hydroxide hydroxide (NaOH; (NaOH; Sigma-Aldrich, Sigma-Aldrich, Buchs, Buchs, Switzerland; Switzerland; cat. no. cat. S8045) no. S8045) •• Milli-Q ultrapure water (from Millipore water system) Milli-Q ultrapure water (from Millipore water system) •• Deionized water from in-house system • Deionized water from in-house system • H. salinarum strain S9 [65] • H.H. salinarum strainstrain L33 S9 [53,66] [65] • • H.Novobiocin salinarum sodiumstrain salt L33 (Sigma-Aldrich, [53,66] Buchs, Switzerland; cat. no. N1628) •• NovobiocinHydrochloric sodium acid (HCl) salt fuming, (Sigma-Aldrich, ≥37% (Sigma-Aldrich, Buchs, Switzerland; Buchs, Switzerland; cat. no. N1628) cat. no. 30721) • ‘CAUTION’ Highly corrosive. Work in a fume hood and wear goggles and protective clothing. Hydrochloric acid (HCl) fuming, 37% (Sigma-Aldrich, Buchs, Switzerland; cat. no. 30721) •• Polyethylene glycol 600 (PEG600; Sigma-Aldr≥ ich, Buchs, Switzerland; cat. no. 87333) ‘CAUTION’ Highly corrosive. Work in a fume hood and wear goggles and protective clothing. • Sucrose (Sigma-Aldrich, Buchs, Switzerland; cat. no. S7903) • Polyethylene glycol 600 (PEG600;2 Sigma-Aldrich, Buchs, Switzerland; cat. no. 87333) • Calcium chloride dihydrate (CaCl ; Merck, Zug, Switzerland; cat. no. 102382) • SucroseDeoxyribonuclease (Sigma-Aldrich, I from bovine Buchs, pancreas Switzerland; (DNase; cat. Sigma-Aldrich, no. S7903) Buchs, Switzerland; cat. no. • CalciumDN25) chloride dihydrate (CaCl2; Merck, Zug, Switzerland; cat. no. 102382) •• 3 DeoxyribonucleaseSodium azide (NaN ; IAppliChem, from bovine Bioswisstec, pancreas Schaffhausen, (DNase; Sigma-Aldrich, Switzerland; cat. Buchs, no. A1430) Switzerland; • ‘CAUTION’ Highly toxic substance. Needs special handling and waste disposal. cat. no. DN25) • Coomassie Brilliant blue R-250 (AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. Sodium azide (NaN ; AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. A1430) • A1092) 3 • ‘CAUTION’Immobilon-P Highlypolyvinylidene toxic substance. difluoride Needs(PVDF) specialtransfer handlingmembrane and (Merck waste Millipore disposal. Ltd., Zug, CoomassieSwitzerland; Brilliant cat. no. blueIPVH00010) R-250 (AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. A1092) • • Immobilon-PAlbumin from polyvinylidenebovine serum (BSA, difluoride Sigma-Aldrich, (PVDF) Buchs, transfer Switzerland; membrane cat. (Merckno. A3912) Millipore Ltd., Zug, •• Switzerland;Tween 20 (AppliChem, cat. no. IPVH00010) Bioswisstec, Schaffhausen, Switzerland; cat. no. A4974) • Anti-penta-His mouse primary antibody (Qiagen, Hombrechtikon, Switzerland; cat. no. 34660) Albumin from bovine serum (BSA, Sigma-Aldrich, Buchs, Switzerland; cat. no. A3912) •• Goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad, Tween 20 (AppliChem, Bioswisstec, Schaffhausen, Switzerland; cat. no. A4974) • Cressier, Switzerland; cat. no. 172-1011) • Anti-penta-HisAmersham ECL Western mouse primaryBlotting Detection antibody Reagents (Qiagen, (GE Hombrechtikon, Healthcare, Opfikon, Switzerland; Switzerland; cat. cat. no. 34660) • Goatno. RPN2209) anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad, Cressier, • • Switzerland;Super RX-N Fuji cat. Medical no. 172-1011) X-ray film (Fujifilm, Dielsdorf, Switzerland; cat. no. 47,410 19284) • AmershamGlycerol (Sigma-Aldrich, ECL Western Buchs, Blotting Switzerland; Detection cat. no. Reagents 49770) (GE Healthcare, Opfikon, Switzerland; •• 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) dissolved in chloroform (Avanti Polar cat. no. RPN2209) Lipids, Alabaster, AL, USA; cat. no. 850375) • Nitrogen (or any other inert) gas

Methods Protoc. 2020, 3, 51 5 of 22

Super RX-N Fuji Medical X-ray film (Fujifilm, Dielsdorf, Switzerland; cat. no. 47,410 19284) • Glycerol (Sigma-Aldrich, Buchs, Switzerland; cat. no. 49770) • 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) dissolved in chloroform (Avanti Polar Lipids, • Alabaster, AL, USA; cat. no. 850375) Nitrogen (or any other inert) gas • n-octyl-β-D-glucopyranoside (OG, Glycon Biochemicals, Luckenwalde, Germany; cat. no. D97001) • Potassium phosphate monobasic (KH PO , Sigma-Aldrich, Buchs, Switzerland; cat. no. P5655) • 2 4 Potassium phosphate dibasic (K HPO , Sigma-Aldrich, Buchs, Switzerland; cat. no. P3786) • 2 4 Uranyl formate (Thomas Scientific, cat. no. C993L42). ‘CAUTION’ Highly toxic substance. • Needs special handling and waste disposal according to radioactivity regulations.

2.2. Equipment PCR cycler: we use a SensoQuest Labcycler • Agarose gel electrophoresis apparatus • pH meter: we use a Seven Compact pH meter (Mettler Toledo, cat. no. 30019028) • Filters for media sterilization: we use Jet Biofil Filter Upper Cups 0.22 µm (Axonlab, • cat. no. FPE-214-000) Incubator for liquid cultures: we use an Infors HT Multitron Standard incubator • Spectrophotometer for liquid cultures: we use a Pharmacia Biotech Ultraspec 3000 • UV-visible spectrophotometer Light microscope: we use a Leitz Laborlux 11 light microscope • Incubator for agar plates: we use a Memmert TNB400 incubator • Centrifuge for 15 mL and 50 mL conical vials: we use a Beckman Coulter Allegra X-15R centrifuge • Centrifuge for 1.5 mL and 2 mL test tubes: we use an Eppendorf Centrifuge 5424R • Ultracentrifuge for 1 mL tubes: we use a Beckman Coulter OptimaTM MAX-XP ultracentrifuge • For membrane homogenization we use a glass homogenizer size 19 (1 mL) from Kontes Glass Co • For chromophore-based protein quantification we use a NanoDrop 1000 spectrophotometer • (Thermo Fisher, Reinach, Switzerland) SDS-PAGE apparatus: we use a Bio-Rad Mini-PROTEAN Tetra System • Blotting apparatus for Western blot: we use a Bio-Rad TransBlot SD Semi-Dry Transfer Cell • Centrifuge for 1 L bottles: we use a Herolab HiCen XL centrifuge • Ultracentrifuge for 94 mL tubes: we use a Beckman Coulter OptimaTM L-90K ultracentrifuge • 10 mL round bottom flasks with glass stoppers and suitable holder • Desiccator equipped with a vacuum pump: we use a Nalgene plastic desiccator (Thermo Fisher, • Reinach, Switzerland) Thermoshaker for 1–2 mL sample tubes: we use an Eppendorf ThermoMixer C • Extruder with heating block and two gas tight syringes: we use an Avanti Mini-Extruder (Avanti cat. • no. 610000) and two 1 mL gas tight syringes (Avanti, cat. no. 610017) Membranes for extruder: we use polycarbonate membranes with pore size 0.2 µm (Avanti, • cat. no. 610006) Filter supports for extruder: we use 10 mm filter supports (Avanti, cat. no. 610014) • Dialysis tubing: we use 10 mm Membra-Cel dialysis tubing with molecular weight cut-off 14 kDa • (Membra-Cel, Roth, cat no. 1780.1) Plastic clips: we use Spectrum Dialysis Tubing Closures (Spectra/Por, cat. no. 888-11614) • 2 L beaker or Erlenmeyer flask • Unautoclaved 2 mL sample tubes • Magnetic stirrer and magnetic stir bar • Methods Protoc. 2020, 3, 51 6 of 22

Temperature regulated water bath with connective tubing and a glass cooling cell: we use a Julabo • F10 ultrapure water bath (Gemini, cat. no. 01819) and a SONOPULS KG 3 (Faust, cat. no. 9.650 235) glass cooling cell with water jacket Micro pH electrode with integrated temperature sensor: we use an InLab Micro Pro-ISM electrode • (Mettler Toledo, cat. no. 51344163) Computer with LabX direct pH 2.3 software (Mettler Toledo) • Warm white LED lamp: we use a JANSÖ LED lamp (IKEA, cat. no. 903.860.12) • Laboratory film: we use Parafilm M sealing film • Syringe and filter: we use disposable 10 mL syringes ONCE/CODAN (HUBERLAB, cat. no. • 3.7410.06) and single use hydrophilic 0.2 µm Sartorius Minisart syringe filters (fisher scientific, cat. no. 10730792) Carbon-coated copper electron microscopy grids: we use 400 mesh copper grids with a thin • carbon film (Gridtech, cat. no. Cu-400CN) Microscope slide • Glow discharge system including a high voltage power supply and a vacuum chamber: we use a • custom build but any commercially available system works Precision forceps: we use 0.1 0.06 mm Dumont Dumoxel Tweezers #5 (World Precision • × Instruments, cat. no. 14098) Blotting paper for microscope slides: we use Macherey-Nagel MN 224 blotting paper for • microscope slides Electron microscopy grid storage box: we use a grid storage box for 100 grids from Electron • Microscopy Sciences Transmission electron microscope: we use a FEI Tecnai Spirit transmission electron microscope • (Thermo Fisher, Reinach, Switzerland) equipped with an FEI Eagle CCD camera

3. Procedure

3.1. Site-Directed Mutagenesis of Bop in pUC18 (0.5 Day) Introduce a C-terminal His -tag into the bop gene, which encodes wild-type BR (BR-wt), by PCR • 10 with pUC18-bop as a template, 50-pUC-BamHI-BR as a forward primer and 30-BR-C-His10-HindIII as a reverse primer (see Section 2.1). You can use a PCR kit of your choice, we use the Phusion High-Fidelity PCR kit. Prepare the following PCR master mix (Table1) on ice or follow the manufacturer’s instructions for your preferred kit:

Table 1. PCR reaction mixture for site-directed mutagenesis of the bop gene.

Component Amount (µL) Final Concentration pUC18-bop plasmid template 1 2 ng/µL (100 ng/µL) 5 -pUC-BamHI-BR forward primer 0 1 2 pM (100 pM) 3 -BR-C-His -HindIII reverse primer 0 10 1 2 pM (100 pM) dNTP’s (10 mM for each dNTP) 1 200 µM Phusion CG buﬀer (5X) 10 1X DMSO (100%) 1.5 3% Phusion DNA Polymerase (2 U/µL) 0.5 0.02 U/µL Nuclease-free water 34 Total 50 Methods Protoc. 2020, 3, 51 7 of 22

Run the PCR reaction using the following program (Table2): •

Table 2. PCR program for site-directed mutagenesis of the bop gene.

Cycles Denaturation (94 ◦C) Annealing (63 ◦C) Elongation (72 ◦C) 1 2 min 32 45 s 45 s 1 min 1 10 min

Purify your PCR product (bop-C-His ) using a PCR puriﬁcation kit (see Section 2.1) and follow • 10 the manufacturer’s instructions. We use an elution volume of 32 µL.

3.2. Insertion of Bop-C-His10 into pHS Blue (0.5 Day) Digest the puriﬁed PCR product with BamHI-HF and HindIII-HF. Prepare the following reaction • mixture (Table3) and incubate for 1.5 h at 37 ◦C.

Table 3. Reaction mixture for the digestion of the PCR product.

Component Amount (µL) Final Concentration Puriﬁed PCR product (350 ng/µL) 20 140 ng/µL CutSmart buﬀer (10X) 5 1X HindIII-HF (20 U/µL) 1 0.4 U/µL BamHI-HF (20 U/µL) 1 0.4 U/µL Nuclease-free water 23 Total 50

In parallel, digest the pHS blue plasmid with BamHI-HF and HindIII-HF as well. Prepare the • following reaction mixture (Table4) and incubate for 1.5 h at 37 ◦C.

Table 4. Reaction mixture for the digestion of the pHS blue plasmid.

Component Amount (µL) Final Concentration pHS blue plasmid (2 µg/µL) 7 280 ng/µL CutSmart buﬀer (10X) 5 1X HindIII-HF (20 U/µL) 3 1.2 U/µL BamHI-HF (20 U/µL) 3 1.2 U/µL Nuclease-free water 32 Total 50

Load the digested insert and the digested pHS blue plasmid on a 1% (w/v) agarose gel prepared • with TAE buﬀer and let it run. Cut out the resulting bands for the insert and the plasmid and purify them using a gel extraction • kit (see Section 2.1). Elute with a volume of 20 µL. Ligate the plasmid and the insert using a DNA ligation kit of your choice (see Section 2.1). • Prepare the following ligation reaction mixture (Table5) and incubate it for 45 min at RT. Methods Protoc. 2020, 3, 51 8 of 22

Table 5. Reaction mixture for the ligation of pHS blue and the insert.

Component Amount (µL) Final Concentration Digested pHS blue plasmid (130 ng/µL) 1 6.5 ng/µL Digested insert (32 ng/µL) 14 22.5 ng/µL Rapid ligation buﬀer (5X) 4 1X T4 DNA ligase (5 U/µL) 1 0.25 U/µL Total 20

3.3. Ampliﬁcation of pHS Blue-Bop-C-His10 in E. coli DH5α (1 Week) Let 50 µL of E. coli DH5α competent cells thaw on ice. • Add 5 µL of the ligation product (pHS blue plasmid with bop-C-His insert) to the competent • 10 cells. Incubate on ice for 20 min. Incubate for 60 s at 42 C. • ◦ Incubate for 2 min on ice. • Add 900 µL of LB and incubate at 700 rpm for 30 min at 37 C in an Eppendorf tube shaker. • ◦ Centrifuge at 14,000 rpm for 2 min at RT. • Remove 880 µL of the supernatant. • Resuspend the pellet in the remaining supernatant. • Spread out the transformed cells on an LB-agar plate supplemented with 25 µg/mL kanamycin. • Incubate overnight at 37 ◦C. Pick 4 colonies from the LB-agar plate and, with each, inoculate 10 mL of LB medium supplemented • with 25 µg/mL kanamycin. Incubate at 180 rpm overnight at 37 ◦C. Use 5 mL of the overnight cultures to prepare glycerol stocks for each colony at a ﬁnal concentration • of 20% glycerol. Isolate the pHS blue-bop-C-His plasmid from the remaining 5 mL of the overnight cultures of • 10 each colony using a plasmid DNA isolation kit of your choice. We use an E.Z.N.A. Plasmid DNA Mini Kit I with an elution volume of 60 µL. Measure the concentration of the isolated pHS blue plasmid by UV-Vis spectroscopy at 260 nm. • The OD260/OD280 ratio should be between 1.8 and 2.0, indicating pure DNA. Digest the isolated plasmids with BamHI-HF and HindIII-HF. Prepare the following reaction • mixture (Table6) and incubate for 1.5 h at 37 ◦C.

Table 6. Reaction mixture for the digestion of the ampliﬁed plasmid.

Component Amount (µL) Final Concentration

Isolated pHS blue-bop-C-His10 (125 ng/µL) 8 100 ng/µL CutSmart buﬀer (10X) 1 1X HindIII-HF (20 U/µL) 0.5 1 U/µL BamHI-HF (20 U/µL) 0.5 1 U/µL Total 10

Load the digested plasmid on a 0.8% (w/v) agarose gel prepared with TAE buﬀer and run the gel. • The gel should show two bands, one for the vector (~10.7 kb) and one for the insert (~1.2 kb). • If that is the case, let the DNA of bacteriorhodopsin in the pHS blue-bop-C-His10 construct be sequenced by a service company and check for your desired modiﬁcation/mutation, and possible random mutations. If the sequence of a clone is correct, inoculate 50 mL LB supplemented with Methods Protoc. 2020, 3, x FOR PEER REVIEW 8 of 21

• Spread out the transformed cells on an LB-agar plate supplemented with 25 µg/mL kanamycin. Incubate overnight at 37 °C. • Pick 4 colonies from the LB-agar plate and, with each, inoculate 10 mL of LB medium supplemented with 25 µg/mL kanamycin. Incubate at 180 rpm overnight at 37 °C. • Use 5 mL of the overnight cultures to prepare glycerol stocks for each colony at a final concentration of 20% glycerol. • Isolate the pHS blue-bop-C-His10 plasmid from the remaining 5 mL of the overnight cultures of each colony using a plasmid DNA isolation kit of your choice. We use an E.Z.N.A. Plasmid DNA Mini Kit I with an elution volume of 60 µL. • Measure the concentration of the isolated pHS blue plasmid by UV-Vis spectroscopy at 260 nm. The OD260/OD280 ratio should be between 1.8 and 2.0, indicating pure DNA. • Digest the isolated plasmids with BamHI-HF and HindIII-HF. Prepare the following reaction mixture (Table 6) and incubate for 1.5 h at 37 °C.

Table 6. Reaction mixture for the digestion of the amplified plasmid.

Component Amount (µL) Final Concentration Isolated pHS blue-bop-C-His10 8 100 ng/µL (125 ng/µL) CutSmart buffer (10X) 1 1X HindIII-HF (20 U/µL) 0.5 1 U/µL BamHI-HF (20 U/µL) 0.5 1 U/µL Total 10

• Load the digested plasmid on a 0.8% (w/v) agarose gel prepared with TAE buffer and run the gel. • The gel should show two bands, one for the vector (~10.7 kb) and one for the insert (~1.2 kb). If Methods Protoc. 2020, 3, 51 9 of 22 that is the case, let the DNA of bacteriorhodopsin in the pHS blue-bop-C-His10 construct be sequenced by a service company and check for your desired modification/mutation, and 25possibleµg/mL kanamycinrandom mutations. with 5 µL glycerolIf the sequence stock of the of corresponding a clone is correct, clone and inoculate incubate 50 at 180mL rpm LB supplemented with 25 µg/mL kanamycin with 5 µL glycerol stock of the corresponding clone overnight at 37 ◦C. and incubate at 180 rpm overnight at 37 °C. Isolate the pHS blue-bop-C-His10 plasmid from the overnight culture using a plasmid DNA •• Isolate the pHS blue-bop-C-His10 plasmid from the overnight culture using a plasmid DNA isolation kit of your choice. We use an E.Z.N.A. Plasmid DNA Midi Kit I with an elution volume isolation kit of your choice. We use an E.Z.N.A. Plasmid DNA Midi Kit I with an elution volume of 500 µL. Measure the DNA concentration as described above. of 500 µL. Measure the DNA concentration as described above. PAUSE STEPSTEP pHSpHS blue-blue-bop-C-Hisbop-C-His10 plasmids can be stored at −2020 °CC for for years. years. 10 − ◦ 3.4.3.4. PreparationPreparation ofof H.H. salinarumSalinarum L33 L33 Cells Cells for for Transformation Transformation (2–3 (2–3 Days) Days) • InoculateInoculate 10 10 mL mL L37 L37 medium medium in in a a 5050 mLmL conicalconical tubetube withwith 1010 µµLL H.H. salinarumsalinarum L33L33 fromfrom aa glycerolglycerol • stock.stock. ThisThis susufficesﬃces forfor fourfour transformations.transformations. • IncubateIncubate the the culture culture at at 170 170 rpm rpm and and 40 40 °CC until until the the OD OD600 isis between 0.60.6 andand 0.8,0.8, typicallytypically reachedreached • ◦ 600 afterafter 2–32–3 days.days.

3.5.3.5. PreparationPreparation ofof PlasmidPlasmid forfor TransformationTransformation (10 (10 Min) Min)

• PlacePlace aa volumevolume containingcontaining 33 µµgg ofof thethe pHS pHS blue- blue-bop-C-Hisbop-C-His1010 plasmidplasmid atat thethe bottombottom ofof aa 1515 mLmL • conicalconical vial,vial, addadd 55 MM NaClNaCl toto thethe dropdrop toto reachreach aa ﬁnalfinal concentrationconcentration ofof 22 MM (e.g.,(e.g., 33µ µLL plasmidplasmid (1(1µ µg/µL)g/µL) + +2 2µ µLL5 5M M NaCl) NaCl)and andmix mix the the resulting resulting drop drop with with a a pipette. pipette. CloseClose thethe tubetube toto avoidavoid evaporationevaporation andand storestore at at room room temperature temperature until until it it is is used used for for transformation. transformation. Methods Protoc. 2020, 3, x FOR PEER REVIEW 9 of 21 3.6.3.6. TransformationTransformation ofof H.H. salinarumsalinarum L33L33 (2(2 H)H) • Pellet the H. salinarum culture at 4000x g for 15 min at room temperature, discard the Pellet the H. salinarum culture at 4000 g for 15 min at room temperature, discard the supernatant, • supernatant, resuspend the pellet in× 1 mL of SPH and take a 50 µL sample for spheroplast resuspend the pellet in 1 mL of SPH and take a 50 µL sample for spheroplast formation control. formation control. Add 50 µL of EDTA solution to the resuspended cells, gently mix by shaking the tube by hand • Add 50 µL of EDTA solution to the resuspended cells, gently mix by shaking the tube by hand and take another 50 µL sample. and take another 50 µL sample. Check the formation of spheroplasts by comparing the 50 µL samples before and after EDTA • Check the formation of spheroplasts by comparing the 50 µL samples before and after EDTA addition in a light microscope. Normal H. salinarum L33 cells are motile rods (Figure1a), while addition in a light microscope. Normal H. salinarum L33 cells are motile rods (Figure 1a), while spheroplasts are round and only move by Brownian motion (Figure1b). spheroplasts are round and only move by Brownian motion (Figure 1b).

Figure 1. H.H. salinarum salinarum L33L33 cells cells as as rods rods and and spheroplasts. spheroplasts. Micrographs Micrographs of H.H. salinarum salinarum L33 cells before (a) and after supplementation of ethylenediaminetetraaceticethylenediaminetetraacetic acid acid (EDTA) (EDTA) to a ﬁnalfinal concentration of 25 mM ( b). ( (aa)) Before Before the the addition addition of of EDTA, EDTA, the the cells cells are are motile motile rods rods up up to to a a length length of of 6 6 µm.µm. Rarely, Rarely, mitotic cells with spherical shape can be found. ( (bb)) After After the the addition addition of of EDTA, EDTA, the archaeal cell envelope, the S-layer, is decomposed and all cells adopt a spherical shape in which they are deprived of autonomous motion. Shown are excerpts of 50 × 5050 µm.µm. × • If all cells turned from rods to spheroplasts, add 200 µL of the resuspended cells to the 15 mL conical vial containing the plasmid and incubate for 20 min at room temperature. • Carefully add an equal volume of PEG solution to the spheroplast-DNA-mix (200 µL spheroplasts + volume of diluted DNA, e.g., 5 µL) and incubate for 20 min at room temperature. • Fill up the conical vial to 10 mL with spheroplast dilution solution and incubate for 30 min at 40 °C.

3.7. Outgrowth of Transformed Cells (2 Days) • Pellet the cells at 4000x g for 10 min at room temperature and resuspend in 1 mL regeneration medium. Place the tube horizontally in the incubator instead of standing in a rack and incubate at 180 rpm and 37 °C for 2 days.

3.8. Plating and Colony Formation (1–2 Weeks) • Dilute the transformed cells and spread out 50 µL on L37-agar plates supplemented with 0.3 µg/mL novobiocin. As transformation efficiency varies strongly, try different dilutions (e.g., 1:100 and 1:1000). Seal the plates in plastic bags or with plastic tape to avoid evaporation during incubation (see 5.2 Equipment Setup). Incubate at 37 °C for 1–2 weeks.

3.9. Colony Picking and Cultivation (1 Week) • Pick 10–20 colonies from the L37-agar plates (Figure 2) and with each, inoculate 20 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 50 mL conical vial. Incubate at 170 rpm and 40 °C for 1 week.

Methods Protoc. 2020, 3, 51 10 of 22

If all cells turned from rods to spheroplasts, add 200 µL of the resuspended cells to the 15 mL • conical vial containing the plasmid and incubate for 20 min at room temperature. Carefully add an equal volume of PEG solution to the spheroplast-DNA-mix (200 µL spheroplasts • + volume of diluted DNA, e.g., 5 µL) and incubate for 20 min at room temperature. Fill up the conical vial to 10 mL with spheroplast dilution solution and incubate for 30 min at • 40 ◦C.

3.7. Outgrowth of Transformed Cells (2 Days) Pellet the cells at 4000 g for 10 min at room temperature and resuspend in 1 mL regeneration • × medium. Place the tube horizontally in the incubator instead of standing in a rack and incubate at 180 rpm and 37 ◦C for 2 days.

3.8. Plating and Colony Formation (1–2 Weeks) Dilute the transformed cells and spread out 50 µL on L37-agar plates supplemented with 0.3 µg/mL • novobiocin. As transformation eﬃciency varies strongly, try diﬀerent dilutions (e.g., 1:100 and 1:1000). Seal the plates in plastic bags or with plastic tape to avoid evaporation during incubation (see Section 5.2). Incubate at 37 ◦C for 1–2 weeks.

3.9. Colony Picking and Cultivation (1 Week) Pick 10–20 colonies from the L37-agar plates (Figure2) and with each, inoculate 20 mL of L37 • supplemented with 0.3 µg/mL novobiocin in a 50 mL conical vial. Incubate at 170 rpm and 40 ◦C Methods Protoc. 2020, 3, x FOR PEER REVIEW 10 of 21 for 1 week. Methods Protoc. 2020, 3, x FOR PEER REVIEW 10 of 21 CRITICALCRITICAL STEPSTEP TryTry toto pickpick coloniescolonies of of di differentfferent sizes sizes and and colour colour intensities. intensities. Colonies Colonies with with a stronga strong CRITICAL red red colour colour STEP may may Try express express to pick considerable considerable colonies of different amounts amounts sizes of of BR-wt. BR-wt. and colour InIn ourour intensities. specificspecific case,case, Colonies thethe colours with didadid strong notnot varyvary red colour significantlysignificantly may express andand thethe considerable sizesize ofof thethe clonescl amouones didntsdid ofnotnot BR-wt. correlatecorrelate In our withwith specific thethe ratioratio case, ofof BR-C-HistheBR-C-His colours1010 todidto BR-wt.BR-wt. not vary significantly and the size of the clones did not correlate with the ratio of BR-C-His10 to BR-wt.

Figure 2. L37-agar plate of H. salinarum L33 transformed with BR-C-His 10. A total of 50 µL of H.

Figuresalinarum 2. L33L37-agarL37-agar cells transformedplate plate of of H.H. salinarumwith salinarum BR-C-His L33L33 10transformed transformedwas plated outwith with after BR-C-His BR-C-His 2 days 10of.10 Aoutgrowth. total A total of 50in of aµL 50 dilution µofL H. of salinarumH.of 1:100 salinarum and L33 incubatedL33 cells cells transformed transformedat 37 °C withfor twowith BR-C-His weeks. BR-C-His10 Show was10 platednwas is an plated outexcerpt after out of2 daysafterthe plate of 2 daysoutgrowth 4 × of4 cm outgrowth inin size.a dilution in a ofdilution 1:100 and of 1:100 incubated and incubated at 37 °C atfor 37 two◦C forweeks. two weeks.Shown Shownis an excerpt is an excerpt of the plate of the 4 plate × 4 cm 4 in4 size. cm in size. 3.10. Glycerol Stock Preparation (30 Min) × 3.10. Glycerol Glycerol Stock Stock Preparation (30 Min) • Centrifuge 8 mL of each culture at 4000× g for 15 min at room temperature and resuspend in 3.2 • Centrifuge 8 mL of each culture at 4000 g for 15 min at room temperature and resuspend in • CentrifugemL L37. Add 8 mL 800 of µL each of 50% culture (w/ vat) glycerol4000× ×g forsolution 15 min to at reach room a temperaturefinal concentration and resuspend of 10%. Freeze in 3.2 3.2mLwith mLL37. liquid L37. Add nitrogen Add800 µL 800 andofµ 50%L store of ( 50%w /atv) − (glycerol80w/ v°C.) glycerol solution solution to reach to a reach final aconcentration ﬁnal concentration of 10%. ofFreeze 10%. Freeze with liquid nitrogen and store at 80 C. with liquid nitrogen and store at −80 °C.− ◦ 3.11. Clone Screening by Small-Volume Purple Membrane Preparation (0.5 Day) 3.11. Clone Screening by Small-Volume Purple Membrane Preparation (0.5 Day) • Centrifuge 10 mL of each culture at 5000× g for 15 min at 4 °C, resuspend in 1.6 mL basal salt • Centrifugeand transfer 10 the mL sample of each to culture a 2 mL at Eppendorf 5000× g for tube. 15 minCentrifuge at 4 °C, at resuspend 8000× g for in 51.6 min mL at basal 4 °C andsalt anddiscard transfer the supernatant. the sample to a 2 mL Eppendorf tube. Centrifuge at 8000× g for 5 min at 4 °C and • discardAdd 700 the µL supernatant. of 10 mM MgSO 4 supplemented with 30 µg DNAse (=12 µL of a 2.5 mg/mL solution). • AddLyse 700the µLcells of by10 resuspendingmM MgSO4 supplemented with a pipette with until 30 the µg solutionDNAse (=12 is homogeneous. µL of a 2.5 mg/mL solution). Lyse CRITICAL the cells by STEP resuspending Remaining with DNA a pi petteleads until to a theviscous, solution inhomogeneous is homogeneous. purple membrane sample CRITICAL and reduces STEP the Remaining yield. DNA leads to a viscous, inhomogeneous purple membrane • sampleRemove and the reducescell debris the by yield. centrifuging the samples at 4300× g for 1 min at 4 °C, followed by two • Removecentrifugations the cell atdebris 7000× by g centrifugingfor 1 min at the4 °C. samples Transfer at 4300×the supernatant g for 1 min toat a4 °C,new followed tube after by eachtwo centrifugationscentrifugation. at 7000× g for 1 min at 4 °C. Transfer the supernatant to a new tube after each • centrifugation.Transfer the samples to 1 mL ultracentrifuge tubes and ultracentrifuge the membranes at 55,000× • Transferg for 15 min the samplesat 4 °C. Resuspend to 1 mL ultracentrifuge the pellets in tu 70bes0 µL and Milli-Q ultracentrifuge ultrapure th watere membranes with a pipette at 55,000× and grepeat for 15 the min last at centrifugation.4 °C. Resuspend the pellets in 700 µL Milli-Q ultrapure water with a pipette and • repeatResuspend the last the centrifugation. pellets in 700 µL Milli-Q ultrapure water and ultracentrifuge the membranes at • Resuspend60,000× g for the 15 pellets min at in 4 700 °C. µL Resuspend Milli-Q ultrapure the pellets water in 700 and µL ultracentrifuge Milli-Q ultrapure the membranes water with at a 60,000×pipette andg for repeat 15 min the at last 4 °C. centrifugation. Resuspend the pellets in 700 µL Milli-Q ultrapure water with a • pipetteResuspend and therepeat pellets the lastin 25 centrifugation. µL Milli-Q ultrapure water with a pipette. •• ResuspendMeasure the the concentration pellets in 25 of µL bacteriorhodopsin Milli-Q ultrapure in water each with sample a pipette. by UV-Vis spectrometry (see 5.2 • MeasureEquipment the Setup). concentration of bacteriorhodopsin in each sample by UV-Vis spectrometry (see 5.2 Equipment Setup). 3.12. Evaluation of BR Expression by Coomassie Brilliant Blue-Stained SDS-PAGE and Western blot 3.12.analysis Evaluation (0.5 Day) of BR Expression by Coomassie Brilliant Blue-Stained SDS-PAGE and Western blot analysis (0.5 Day) • Load membranes of each sample corresponding to 5 µg of bacteriorhodopsin on a 13.5% SDS- • LoadPAGE membranes gel. As a ofnegative each sample control, corresponding include an to equivalent 5 µg of bacteriorhodopsin of H. salinarum onS9 a membranes13.5% SDS- PAGE gel. As a negative control, include an equivalent of H. salinarum S9 membranes

Methods Protoc. 2020, 3, x FOR PEER REVIEW 10 of 21

CRITICAL STEP Try to pick colonies of different sizes and colour intensities. Colonies with a strong red colour may express considerable amounts of BR-wt. In our specific case, the colours did not vary significantly and the size of the clones did not correlate with the ratio of BR-C-His10 to BR-wt.

Figure 2. L37-agar plate of H. salinarum L33 transformed with BR-C-His10. A total of 50 µL of H. salinarum L33 cells transformed with BR-C-His10 was plated out after 2 days of outgrowth in a dilution of 1:100 and incubated at 37 °C for two weeks. Shown is an excerpt of the plate 4 × 4 cm in size.

3.10. Glycerol Stock Preparation (30 Min) • Centrifuge 8 mL of each culture at 4000× g for 15 min at room temperature and resuspend in 3.2 MethodsmL Protoc. L37.2020 Add, 3, 51800 µL of 50% (w/v) glycerol solution to reach a final concentration of 10%. Freeze11 of 22 with liquid nitrogen and store at −80 °C. 3.11. Clone Screening by Small-Volume Purple Membrane Preparation (0.5 Day) 3.11. Clone Screening by Small-Volume Purple Membrane Preparation (0.5 Day) Centrifuge 10 mL of each culture at 5000 g for 15 min at 4 ◦C, resuspend in 1.6 mL basal salt and • Centrifuge 10 mL of each culture at 5000×× g for 15 min at 4 °C, resuspend in 1.6 mL basal salt transfer the sample to a 2 mL Eppendorf tube. Centrifuge at 8000 g for 5 min at 4 ◦C and discard and transfer the sample to a 2 mL Eppendorf tube. Centrifuge ×at 8000× g for 5 min at 4 °C and the supernatant. discard the supernatant. Add 700 µL of 10 mM MgSO4 supplemented with 30 µg DNAse (=12 µL of a 2.5 mg/mL solution). • Add 700 µL of 10 mM MgSO4 supplemented with 30 µg DNAse (=12 µL of a 2.5 mg/mL solution). Lyse the cells by resuspending with a pipette until the solution is homogeneous. Lyse the cells by resuspending with a pipette until the solution is homogeneous. CRITICAL STEPSTEP RemainingRemaining DNADNA leadsleads toto aa viscous,viscous, inhomogeneousinhomogeneous purplepurple membranemembrane samplesample andand reducesreduces thethe yield.yield. • RemoveRemove the cell debris debris by by centrifuging centrifuging the the samples samples at at4300× 4300 g forg for 1 min 1 min at 4 at °C, 4 followedC, followed by two by • × ◦ twocentrifugations centrifugations at 7000× at 7000 g for g1 formin 1 at min 4 °C. at 4TransferC. Transfer the supernatant the supernatant to a new to a newtube tubeafter aftereach × ◦ eachcentrifugation. centrifugation. • TransferTransfer the the samples samples to to 1 1 mL mL ultracentrifuge ultracentrifuge tubes tubes and and ultracentrifuge ultracentrifuge the th membranese membranes at at 55,000 55,000×g • g for 15 min at 4 °C. Resuspend the pellets in 700 µL Milli-Q ultrapure water with a pipette and× for 15 min at 4 ◦C. Resuspend the pellets in 700 µL Milli-Q ultrapure water with a pipette and repeatrepeat thethe lastlast centrifugation.centrifugation. • ResuspendResuspend thethe pelletspellets inin 700700 µµLL Milli-QMilli-Q ultrapureultrapure waterwater andand ultracentrifugeultracentrifuge thethe membranesmembranes atat • 60,00060,000× g for 15 min at 4 °C.C. Resuspend the pellets in 700 µµLL Milli-QMilli-Q ultrapureultrapure waterwater withwith aa × ◦ pipettepipette andand repeatrepeat thethe lastlast centrifugation.centrifugation. • Resuspend the pellets in 25 µL Milli-Q ultrapure water with a pipette. Resuspend the pellets in 25 µL Milli-Q ultrapure water with a pipette. • Measure the concentration of bacteriorhodopsin in each sample by UV-Vis spectrometry (see 5.2 Measure the concentration of bacteriorhodopsin in each sample by UV-Vis spectrometry • Equipment Setup). (see Section 5.2).

3.12. Evaluation of BR Expression byby CoomassieCoomassie BrilliantBrilliant Blue-StainedBlue-Stained SDS-PAGESDS-PAGE and and Western Western blot blot analysis (0.5analysis Day) (0.5 Day) • LoadLoad membranesmembranes of of each each sample sample corresponding corresponding to 5toµ g5 ofµg bacteriorhodopsin of bacteriorhodopsin on a 13.5%on a 13.5% SDS-PAGE SDS- • gel.PAGE As agel. negative As a control,negative include control, an include equivalent an ofequivalentH. salinarum of S9H. membranessalinarum S9 corresponding membranes to 5 µg of bacteriorhodopsin (for expression and purification of S9 membranes see Sections 3.14 and 3.15 of this protocol). If available, as a positive control, include an equivalent of membranes from a H. salinarum strain, expressing a His10-tagged bacteriorhodopsin corresponding to 5 µg of bacteriorhodopsin. Run the electrophoresis at 200 V until the dye front reaches the end of the gel and stain the gel using Coomassie Brilliant blue R-250 according to standard protocols. For Western blot, load an equivalent of membranes of each sample corresponding to 0.5 µg of • bacteriorhodopsin on a 13.5% SDS-PAGE gel. As a negative control, include an equivalent of H. salinarum S9 membranes corresponding to 0.5 µg of bacteriorhodopsin. Run the electrophoresis at 200 V. Transfer the samples onto a PVDF-membrane, e.g., using a semi-dry blotting system. Incubate • the membrane in 30 mL TBS containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation to reduce unspecific binding of the antibodies. Incubate the membrane with anti-penta-His mouse primary antibody diluted 1:3000 in 30 mL TBS • containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation. Wash the membrane three times with 30 mL TBS containing 0.5% (v/v) Tween 20 for 10 min at • room temperature under gentle agitation. Incubate with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:3000 in 30 mL TBS containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation. Wash the membrane three times with 30 mL TBS containing 0.5% (v/v) Tween 20 for 10 min at • room temperature under gentle agitation. Add 2 mL of ECL-detection reagent (1 mL of each) to the membrane and incubate for 2 min at • room temperature under gentle agitation. Seal the membrane in a plastic bag and expose an X-ray film. Methods Protoc. 2020, 3, x FOR PEER REVIEW 11 of 21

corresponding to 5 µg of bacteriorhodopsin (for expression and purification of S9 membranes see sections 3.14 and 3.15 of this protocol). If available, as a positive control, include an equivalent of membranes from a H. salinarum strain, expressing a His10-tagged bacteriorhodopsin corresponding to 5 µg of bacteriorhodopsin. Run the electrophoresis at 200 V until the dye front reaches the end of the gel and stain the gel using Coomassie Brilliant blue R- 250 according to standard protocols. • For Western blot, load an equivalent of membranes of each sample corresponding to 0.5 µg of bacteriorhodopsin on a 13.5% SDS-PAGE gel. As a negative control, include an equivalent of H. salinarum S9 membranes corresponding to 0.5 µg of bacteriorhodopsin. Run the electrophoresis at 200 V. • Transfer the samples onto a PVDF-membrane, e.g., using a semi-dry blotting system. Incubate the membrane in 30 mL TBS containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation to reduce unspecific binding of the antibodies. • Incubate the membrane with anti-penta-His mouse primary antibody diluted 1:3000 in 30 mL TBS containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation. • Wash the membrane three times with 30 mL TBS containing 0.5% (v/v) Tween 20 for 10 min at room temperature under gentle agitation. Incubate with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:3000 in 30 mL TBS containing 3% (w/v) BSA for 1 h at room temperature under gentle agitation. • Wash the membrane three times with 30 mL TBS containing 0.5% (v/v) Tween 20 for 10 min at room temperature under gentle agitation. • Methods Add Protoc. 2 mL2020 of, 3 ,ECL-detection 51 reagent (1 mL of each) to the membrane and incubate for 2 min12 of at 22 room temperature under gentle agitation. Seal the membrane in a plastic bag and expose an X- ray film. • Estimate the ratio of His1010 -tagged to wild-type bacteriorhodopsin in the diﬀerent samples by • Estimate the ratio of His -tagged to wild-type bacteriorhodopsin in the different samples by SDS-PAGE (Figure(Figure3 a)3a) and and identify identify the the best best clone(s). clone(s) If. If a purea pure clone clone could could be be obtained obtained (i.e., (i.e., no orno aor very a very small small lower lower band), band), continue continue working working with with the glycerol the glycerol stock stock of the of corresponding the corresponding clone. Asclone. an example,As an example, in Figure in3 Figurea, clone 3a, number clone number 2 (C2) contains 2 (C2) contains a considerable a considerable amount of amount BR-wt, of while BR- clonewt, while number clone 4 (C4)number can 4 be (C4) considered can be considered of high purity. of high The purity. Western The blot Western in Figure blot3b in shows Figure that 3b cloneshows C4 that contains clone theC4 contains engineered the C-terminal engineered His C-terminal10-tag. A smallerHis10-tag. band A smaller around 50band kDa around indicates 50 thekDa presence indicates of the SDS-resistant presence of BR-C-HisSDS-resistant10 dimers. BR-C-His10 dimers.

Figure 3. AnalysisAnalysis of of BR-C-His BR-C-His10 clones10 clones by SDS-PAGE by SDS-PAGE and and Western Western blot. blot. (a) Coomassie (a) Coomassie Brilliant Brilliant blue- stainedblue-stained 13.5% 13.5% SDS/polyacrylamide SDS/polyacrylamide gel of gelisolated of isolated purple purple membranes membranes from H. from salinarumH. salinarum L33 clonesL33

transformedclones transformed with BR-C-His with BR-C-His10 (C1–C8)10 (C1–C8) and BR-wt and (isolated BR-wt (isolated from H. from salinarumH. salinarum S9) as aS9) control. as a control. BR-C- HisBR-C-His10 migrates10 migrates slower slowercompared compared to BR-wt to BR-wtbecause because of the ofadditional the additional His10-tag. His 10In-tag. each Inlane each purple lane purplemembranes membranes corresponding corresponding to 5 µg toof 5bacteriorhodopsinµg of bacteriorhodopsin were loaded. were loaded. (b) Western (b) Western blot analysis blot analysis of BR-

wtof BR-wtand BR-C-His and BR-C-His10 (C4)10 performed(C4) performed using usingan SDS an gel SDS run gel under run under similar similar conditions conditions as in as(a). in The (a). Theprimary primary and andsecondary secondary antibodies antibodies used used were were mous mousee monoclonal monoclonal anti-penta-H anti-penta-Hisis antibody antibody (dilution 1:3000) andand horseradish horseradish peroxidase-conjugated peroxidase-conjugated goat anti-mousegoat anti-mouse antibody antibody (dilution 1:3000),(dilution respectively. 1:3000), respectively.BR-wt showed BR-wt no band, showed due tono theband, absence due to of the a His absence10-tag. BR-C-Hisof a His1010-tag.reacted BR-C-His with10 the reacted anti-penta-His with the antibody, conﬁrming the presence of the introduced His10-tag. A weaker band around 50 kDa indicates

the presence of SDS-resistant BR-C-His10 dimers.

3.13. Iterative Clone-Picking to Optimize Expression of Engineered BR (2–3 Weeks) If the ratio of His -tagged to wild-type bacteriorhodopsin of the best clone is not satisfactory, • 10 inoculate 20 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 50 mL conical vial with 10 µL of the glycerol stock of the best clone and incubate at 170 rpm for 1 week at 40 ◦C. Prepare from this culture new L37-agar plates, pick clones and repeat the analysis as described in Sections 3.8–3.12. This process may have to be iterated several times to obtain a clone that only expresses the His10-tagged bacteriorhodopsin construct.

3.14. Large-Volume Cultivation of a Selected Transformed H. Salinarum L33 Clone (2 Weeks) Inoculate 50 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 250 mL Erlenmeyer flask • with 50 µL of the glycerol stock of the selected clone. Incubate at 170 rpm for 1 week at 40 ◦C. Inoculate 2 L of L37 supplemented with 0.3 µg/mL novobiocin in a 5 L Erlenmeyer flask with 40 • mL of the previous culture. Incubate at 170 rpm for 1 week at 40 ◦C. For a standard large-volume cultivation, we inoculate three flasks containing 2 L of each medium. Methods Protoc. 2020, 3, x FOR PEER REVIEW 12 of 21

anti-penta-His antibody, confirming the presence of the introduced His10-tag. A weaker band around

50 kDa indicates the presence of SDS-resistant BR-C-His10 dimers.

3.13. Iterative Clone-Picking to Optimize Expression of Engineered BR (2–3 Weeks)

• If the ratio of His10-tagged to wild-type bacteriorhodopsin of the best clone is not satisfactory, inoculate 20 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 50 mL conical vial with 10 µL of the glycerol stock of the best clone and incubate at 170 rpm for 1 week at 40 °C. Prepare from this culture new L37-agar plates, pick clones and repeat the analysis as described in sections 3.8 to 3.12. This process may have to be iterated several times to obtain a clone that only expresses Methodsthe Protoc. His10 2020-tagged, 3, x FOR bacteriorhodopsin PEER REVIEW construct. 12 of 21

3.14. anti-penta-HisLarge-Volume antibody,Cultivation confirming of a Selected the presenceTransformed of the H. introduced Salinarum His L3310-tag. Clone A weaker(2 Weeks) band around 50 kDa indicates the presence of SDS-resistant BR-C-His10 dimers. • Inoculate 50 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 250 mL Erlenmeyer flask 3.13. withIterative 50 µL Clone-Picking of the glycerol to Optimize stock of Expression the selected of Engineeredclone. Incubate BR (2–3 at 170Weeks) rpm for 1 week at 40 °C. • Inoculate 2 L of L37 supplemented with 0.3 µg/mL novobiocin in a 5 L Erlenmeyer flask with 40 • If the ratio of His10-tagged to wild-type bacteriorhodopsin of the best clone is not satisfactory, mL of the previous culture. Incubate at 170 rpm for 1 week at 40 °C. For a standard large-volume inoculate 20 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 50 mL conical vial with cultivation, we inoculate three flasks containing 2 L of each medium. 10 µL of the glycerol stock of the best clone and incubate at 170 rpm for 1 week at 40 °C. Prepare 3.15. fromPurple this Membrane culture newPreparation L37-agar (1.5 plates, Days) pick clones and repeat the analysis as described in sections 3.8 to 3.12. This process may have to be iterated several times to obtain a clone that only expresses • Centrifuge the culture at 10,000× g for 20 min at 4 °C, resuspend the pellet in 400 mL basal salt the His10-tagged bacteriorhodopsin construct. using a 25 mL serological pipette and repeat the last centrifugation. Before centrifugation, 3.14. measureLarge-Volume the weight Cultivation of the of empty a Selected centrifuga Transformedtion tubes H. Salinarum to determine L33 Clone the cell (2 Weeks) mass. • Resuspend the pellet in 25 mL/g cell mass of Milli-Q ultrapurified water supplemented with • Inoculate 50 mL of L37 supplemented with 0.3 µg/mL novobiocin in a 250 mL Erlenmeyer flask 0.02% (w/v) NaN3 (preservative to inhibit bacterial growth). Add 1 mg DNase/g cell mass and with 50 µL of the glycerol stock of the selected clone. Incubate at 170 rpm for 1 week at 40 °C. gently stir the solution at 4 °C overnight to lyse the cells by osmotic shock. Methods• Inoculate Protoc. 2020 2, L3, of 51 L37 supplemented with 0.3 µg/mL novobiocin in a 5 L Erlenmeyer flask with13 of 40 22 CRITICAL STEP Remaining DNA leads to a viscous, inhomogeneous purple membrane mL of the previous culture. Incubate at 170 rpm for 1 week at 40 °C. For a standard large-volume sample and reduces the yield. cultivation, we inoculate three flasks containing 2 L of each medium. 3.15.• Remove Purple Membrane the cell debris Preparation by centrifuging (1.5 Days) the sample at 4300× g for 10 min at 4 °C, followed by two centrifugations at 7600× g for 10 min at 4 °C. Transfer the supernatant to a new tube after each 3.15.Centrifuge Purple Membrane the culture Preparation at 10,000 (1.5 Days)g for 20 min at 4 ◦C, resuspend the pellet in 400 mL basal salt • × usingcentrifugation. a 25 mL serological Over the course pipette of and the repeat three thecentri lastfugations, centrifugation. the cell Before debris centrifugation, pellets should measure change • Centrifuge the culture at 10,000× g for 20 min at 4 °C, resuspend the pellet in 400 mL basal salt thein color weight from of thebrown empty to purple. centrifugation tubes to determine the cell mass. • Transferusing a 25the mL supernatant serological topipette two 94and mL repeat ultracen the trifugelast centrifugation. tubes, fill them Before up centrifugation, with Milli-Q Resuspend the pellet in 25 mL/g cell mass of Milli-Q ultrapurified water supplemented with 0.02% • ultrapurifiedmeasure the weight water and of the ultracentrifuge empty centrifuga at 55,000×tion tubes g for to1 h determine at 4 °C. Resuspend the cell mass. the pellets in Milli- (w/v) NaN3 (preservative to inhibit bacterial growth). Add 1 mg DNase/g cell mass and gently stir • QResuspend ultrapurified the pelletwater inwith 25 amL/g pipette, cell combine mass of themMilli-Q in oneultrapurified 94 mL ultracentrifuge water supplemented tube, fill itwith up the solution at 4 ◦C overnight to lyse the cells by osmotic shock. with0.02% Milli-Q (w/v) NaN ultrapurified3 (preservative water toand inhibit repeat bacterial the last grcentrifugation.owth). Add 1 mg DNase/g cell mass and gently CRITICAL stir the solution STEP ThereRemaining at 4 °C might overniDNA stillght be leads to some lyse to remainingthe a viscous, cells by cell osmotic inhomogeneous debris shock. as a gray purple spot at membrane the center sampleof the CRITICAL pellets. and reduces Resuspend STEP the Remaining yield. the purple DNA parts leads around to a itviscous, very carefully inhomogeneous with a pipette purple by membraneletting the watersample flow and over reduces the pelletthe yield. without detaching the gray part from the wall of the tube. If the gray Remove the cell debris by centrifuging the sample at 4300 g for 10 min at 4 ◦C, followed by two • × centrifugationsspotsRemove are the relatively cell atdebris 7600 small, byg centrifugingthefor 10wh minole pellets at the 4 sampleC. can Transfer be at resuspended 4300× the supernatant g for 10 af termin the toat afirst4 new°C, centrifugation. followed tube after by each two It × ◦ centrifugation.willcentrifugations be easier to Over atseparate 7600× the coursegthe for cell 10 of debrismin the at three after4 °C. centrifugations, the Transfer second the centrifugation supernatant the cell debris whento pelletsa new the tubetwo should grayafter change spots each infromcentrifugation. color the from previous brown Over pellets tothe purple. course are combined of the three in one centri spot.fugations, the cell debris pellets should change • in color from brown to purple. TransferResuspend the supernatantthe pellet in to twoMilli-Q 94 mL ultrapurified ultracentrifuge water tubes, in fillthe them same up tube with Milli-Qwith a ultrapurifiedpipette and • ultracentrifugeTransfer the supernatant at 60,000× g forto 1two h at 94 4 °C.mL Resuspend ultracentrifuge the pellet tubes, with fill Milli-Q them ultrapurified up with Milli-Q water water and ultracentrifuge at 55,000 g for 1 h at 4 ◦C. Resuspend the pellets in Milli-Q ultrapurified ultrapurified water and ultracentrifuge× at 55,000× g for 1 h at 4 °C. Resuspend the pellets in Milli- waterin the withsame atube pipette, and repeat combine the themlast centrifugation. in one 94 mL ultracentrifuge tube, fill it up with Milli-Q • Q ultrapurified water with a pipette, combine them in one 94 mL ultracentrifuge tube, fill it up ultrapurifiedResuspend the water pellet and in repeat~0.5 mL/g the lastcell centrifugation.mass of Milli-Q ultrapurified water supplemented with 0.02%with Milli-Q (w/v) NaN ultrapurified3 with a pipette water and homogenizerepeat the last using centrifugation. a glass homogenizer. You may have to adjust CRITICALCRITICAL the volume STEPSTEP TheredependingThere mightmight on stillstill the bebe somesomeexpressi remainingremainingon efficiency cellcell debrisdebris of asasyour aa graygray construct spot at theand center the ofconcentrationof thethe pellets.pellets. Resuspendneeded for following the purple experiments. parts around it very carefully with a pipettepipette byby lettingletting thethe • waterMeasurewater flowflow the overover concentration thethe pelletpellet without withoutof bacteriorhodopsin detachingdetaching thethe in graygray your partpart sample fromfrom by thethe UV-Vis wallwall ofof spectrometry thethe tube.tube. If the (see gray 5.2 spotsEquipmentspots areare relativelyrelatively Setup). small,small, thethe wholewhole pelletspellets cancan bebe resuspendedresuspended afterafter thethe firstfirst centrifugation.centrifugation. ItIt willwill bebe easiereasier toto separateseparate thethe cellcell debrisdebris afterafter thethe secondsecond centrifugationcentrifugation whenwhen thethe twotwo graygray spots fromfrom thethe previousprevious pelletspellets areare combinedcombined inin oneone spot.spot. • ResuspendResuspend thethe pelletpellet inin Milli-QMilli-Q ultrapurifiedultrapurified water in thethe samesame tubetube withwith aa pipettepipette andand • ultracentrifugeultracentrifuge at 60,000× 60,000 g gforfor 1 h 1 at h 4 at °C. 4 ResuspendC. Resuspend the pellet the pellet with withMilli-Q Milli-Q ultrapurified ultrapurified water × ◦ waterin the insame the tube same and tube repeat and repeat the last the centrifugation. last centrifugation. • ResuspendResuspend thethe pelletpellet inin ~0.5~0.5 mLmL/g/g cellcell massmass ofof Milli-QMilli-Q ultrapurifiedultrapurified waterwater supplementedsupplemented withwith • 0.02% (w/v) NaN3 with a pipette and homogenize using a glass homogenizer. You may have to 0.02% (w/v) NaN3 with a pipette and homogenize using a glass homogenizer. You may have to adjustadjust thethe volume volume depending depending on the on expression the expressi efficiencyon efficiency of your construct of your and construct the concentration and the neededconcentration for following needed experiments. for following experiments. • MeasureMeasure the concentration concentration of of bacteriorhodopsin bacteriorhodopsin in in your your sample sample by byUV-Vis UV-Vis spectrometry spectrometry (see (see 5.2 • MethodsSectionEquipment Protoc. 5.22020). , Setup).3, x FOR PEER REVIEW 13 of 21

Determine the purity of your sample and validate the presence of the His10-tag by Coomassie • Determine the purity of your sample and validate the presence of the His10-tag by Coomassie Brilliant blue-stained SDS-PAGE and Western blot (see Section 3.12). Brilliant blue-stained SDS-PAGE and Western blot (see Section 3.12).

PAUSEPAUSE STEPSTEP PurplePurple membranemembrane samplessamples inin 0.02%0.02% NaNNaN33 cancan bebe storedstored atat 44 ◦°CC forfor years.years.

3.16. Detergent-Mediated Reconstitution of BR into PreformedPreformed LiposomesLiposomes (2(2 Days)Days) • DryDry 1010 mgmg ofof DOPCDOPC dissolveddissolved inin chloroformchloroform inin aa 1010 mLmL roundround bottombottom flaskflask underunder aa constantconstant • streamstream ofof nitrogennitrogen gasgas withwith gentlegentle shaking.shaking. A thin layer of lipid is beneficialbeneficial forfor thethe optimaloptimal formationformation ofof liposomes.liposomes. Remove residual chloroformchloroform tracestraces byby keepingkeeping thethe flaskflask underunder vacuumvacuum inin aa desiccatordesiccator overnight.overnight. • OnOn thethe nextnext day, day, solubilize solubilize 400 400µ gµg of of BR BR in 3%in 3% (w /(vw) OG/v) OG at a at protein a protein concentration concentration of 1 mgof 1/mL mg/mL (e.g., • add(e.g., 120 addµ L120 of µL 10% of (10%w/v) ( OGw/v) to OG 100 toµ 100L 4 µL mg /4mL mg/mL BR and BR 180andµ 180L ultrapure µL ultrapure water). water). Incubate Incubate the mixturethe mixture for3 for h at3 h room at room temperature temperature under under gentle gentle rotation rotation or shaking or shaking (avoid (avoid foam foam formation) formation) and ensureand ensure that it that is protected it is protected from light. from Finally, light. the Finally, mixture the is mixture centrifuged is ce forntrifuged 15 min at for 100,000 15 ming toat × remove100,000× unsolubilized g to remove unsolubilized material. material. • In the meantime, add 2 mL of hydration buffer to the dried lipid and shake it for around 30 min until the lipid is completely resuspended. • Assemble the extruder with a 200 nm pore size membrane and one filter support on each side. Flush it with 1 mL of hydration buffer using the syringes to pass the solution back and forth. Then, pass the liposome suspension through in the same way 19 times. With an increasing number of passages, the suspension should become more transparent, indicating a reduction in particle size. • Start this step before the centrifugation of solubilized BR. Destabilize the extruded liposomes with 0.4% (w/v) OG by adding 83 µL of 10% (w/v) OG for 15 min with gentle shaking. • Add the solubilized BR to the destabilized liposomes and incubate with gentle shaking for 30 min. CRITICAL STEP The final OG concentration after adding the solubilized protein to the liposomes should always be close to 0.8%. This ensures optimal destabilization and the most efficient protein insertion. Should you want to change the amount of protein for the reconstitution, make sure to compensate the amount of detergent added. • In the meantime, soak the dialysis tubing and rinse it with Milli-Q ultrapure water. • Close the dialysis tubing on one side with a plastic clip, fill in the protein liposome mixture and then close the other side with a second plastic clip. Place the filled dialysis tube in 2 L of dialysis buffer. Add a magnetic stir bar and dialyse overnight at 4 °C with gentle stirring. Up to two samples can be dialysed together in 2 L of dialysis buffer. • Harvest the dialysed samples and ultracentrifuge them 10 min at 200,000× g. Resuspend the pellet in 1 mL of measuring solution by pipetting and centrifuge again. Wash the samples once more by repeating the resuspension and centrifugation, and finally resuspend the proteoliposomes in 800 µL of measuring solution.

3.17. Functional Characterization of BR Proteoliposomes by Photoactivity Assay (1 Day) • Transfer the proteoliposome suspension to a clear unautoclaved 2 mL tube and add an appropriately sized magnetic stir bar. Autoclaved tubes tend to get cloudy, thus reducing the illumination of the samples in the next step. • The photoactivity of the proteoliposomes is assessed by measuring the pH change in the extravesicular medium upon illumination using a micro pH electrode. The sample should be constantly stirred and cooled to 18 °C by placing the tube in a transparent glass cooling cell with a water jacket. Illumination is provided by a warm white LED lamp (i.e., full spectrum). Data are collected in intervals of 30 s using a computer connected to the pH meter and running an appropriate pH meter software. Optimal illumination cycles for the described proteoliposomes comprise an initial 15 min adaptation period in the dark, followed by alternating periods of illumination and darkness of 15 min each (see Figure 4). Make sure to collect at least 4 peaks as

Methods Protoc. 2020, 3, x FOR PEER REVIEW 13 of 21

• Determine the purity of your sample and validate the presence of the His10-tag by Coomassie Brilliant blue-stained SDS-PAGE and Western blot (see Section 3.12). PAUSE STEP Purple membrane samples in 0.02% NaN3 can be stored at 4 °C for years.

3.16. Detergent-Mediated Reconstitution of BR into Preformed Liposomes (2 Days) • Dry 10 mg of DOPC dissolved in chloroform in a 10 mL round bottom flask under a constant stream of nitrogen gas with gentle shaking. A thin layer of lipid is beneficial for the optimal formation of liposomes. Remove residual chloroform traces by keeping the flask under vacuum in a desiccator overnight. • On the next day, solubilize 400 µg of BR in 3% (w/v) OG at a protein concentration of 1 mg/mL (e.g., add 120 µL of 10% (w/v) OG to 100 µL 4 mg/mL BR and 180 µL ultrapure water). Incubate the mixture for 3 h at room temperature under gentle rotation or shaking (avoid foam formation) and ensure that it is protected from light. Finally, the mixture is centrifuged for 15 min at Methods Protoc. 2020, 3, 51 14 of 22 100,000× g to remove unsolubilized material. • In the meantime, add 2 mL of hydration buffer to the dried lipid and shake it for around 30 min Inuntil the the meantime, lipid is completely add 2 mL of resuspended. hydration bu ffer to the dried lipid and shake it for around 30 min • • untilAssemble the lipid the isextruder completely with resuspended. a 200 nm pore size membrane and one filter support on each side. AssembleFlush it with the 1 extruder mL of hydration with a 200 buffer nm pore using size the membrane syringes to and pass one the filter solution support back on and each forth. side. • FlushThen, itpass with the 1 mL liposome of hydration suspension buffer using through the syringesin the same to pass way the 19 solution times. back With and an forth. increasing Then, passnumber the liposomeof passages, suspension the suspension through should in the become same way more 19 transparent, times. With anindicating increasing a reduction number ofin passages,particle size. the suspension should become more transparent, indicating a reduction in particle size. • StartStart thisthis step step before before the the centrifugation centrifugation of solubilizedof solubilized BR. BR. Destabilize Destabilize the extrudedthe extruded liposomes liposomes with • 0.4%with (0.4%w/v) OG(w/v by) OG adding by adding 83 µL 83 of µL 10% of ( w10%/v) OG(w/v for) OG 15 for min 15 with min gentle with gentle shaking. shaking. • AddAdd thethe solubilized solubilized BR BR to to the the destabilized destabilized liposomes liposomes and and incubate incubate with with gentle gentle shaking shaking for 30 for min. 30 • min. CRITICAL STEPSTEP TheThe finalfinal OGOG concentrationconcentration afterafter addingadding thethe solubilizedsolubilized proteinprotein toto thethe liposomesliposomes shouldshould alwaysalways bebe closeclose toto 0.8%.0.8%. ThisThis ensuresensures optimaloptimal destabilizationdestabilization andand thethe mostmost eefficientfficient proteinprotein insertion. insertion. Should Should you wantyou towant change to thechange amount the of amount protein forof theprotein reconstitution, for the makereconstitution, sure to compensate make sure theto compen amountsate of detergent the amount added. of detergent added. • InIn thethe meantime,meantime, soaksoak thethe dialysisdialysis tubingtubing andand rinserinse itit withwith Milli-QMilli-Q ultrapureultrapure water. water. •• CloseClose thethe dialysisdialysis tubingtubing onon oneone sideside withwith aa plasticplastic clip,clip, fillfill inin thethe proteinprotein liposomeliposome mixturemixture andand • thenthen closeclose thethe otherother sideside withwith aa secondsecond plasticplastic clip.clip. PlacePlace thethe filledfilled dialysisdialysis tubetube inin 22 LL ofof dialysisdialysis buffer. Add a magnetic stir bar and dialyse overnight at 4 °C with gentle stirring. Up to two buffer. Add a magnetic stir bar and dialyse overnight at 4 ◦C with gentle stirring. Up to two samplessamples cancan bebe dialyseddialysed togethertogether inin 22 LL ofof dialysisdialysis bubuffer.ffer. • HarvestHarvest thethe dialysed dialysed samples samples and and ultracentrifuge ultracentrifuge them them 10 min 10 atmin 200,000 at 200,000×g. Resuspend g. Resuspend the pellet the • × inpellet 1 mL in of 1 measuringmL of measuring solution solution by pipetting by pipetting and centrifuge and centrifuge again. Wash again. the Wash samples the oncesamples more once by repeatingmore by therepeating resuspension the andresuspension centrifugation, and and centrifugation, finally resuspend and the finally proteoliposomes resuspend in 800the µproteoliposomesL of measuring solution.in 800 µL of measuring solution.

3.17.3.17. Functional CharacterizationCharacterization of BR ProteoliposomesProteoliposomes byby PhotoactivityPhotoactivity AssayAssay (1(1 Day)Day) • TransferTransfer the the proteoliposome proteoliposome suspension suspension to a clearto a unautoclaved clear unautoclaved 2 mL tube 2 and mL add tube an appropriatelyand add an • sizedappropriately magnetic sized stir bar. magnetic Autoclaved stir bar. tubes Autoclaved tend to get tubes cloudy, tend thus to reducing get clou thedy, illuminationthus reducing of thethe samplesillumination in the of next the samples step. in the next step. • TheThe photoactivityphotoactivity ofof thethe proteoliposomesproteoliposomes isis assessedassessed byby measuringmeasuring thethe pHpH changechange inin thethe • extravesicularextravesicular medium upon illumination illumination using using a a micro micro pH pH electrode. electrode. The The sample sample should should be constantly stirred and cooled to 18 °C by placing the tube in a transparent glass cooling cell with be constantly stirred and cooled to 18 ◦C by placing the tube in a transparent glass cooling cell witha water a water jacket. jacket. Illumination Illumination is provided is provided by a warm by a warm white white LED lamp LED lamp(i.e., full (i.e., spectrum). full spectrum). Data Dataare collected are collected in intervals in intervals of 30 of s 30 using s using a comp a computeruter connected connected to to the the pH pH meter meter and and running anan appropriateappropriate pHpH metermeter software.software. OptimalOptimal illuminationillumination cyclescycles forfor thethe describeddescribed proteoliposomesproteoliposomes comprisecomprise anan initialinitial 1515 minmin adaptationadaptation periodperiod inin thethe dark,dark, followedfollowed byby alternatingalternating periodsperiods ofof illuminationillumination andand darknessdarkness ofof 1515 minmin eacheach (see(see FigureFigure4 4).). Make Make sure sure to to collect collect at at least least 4 4 peaks peaks as as the ﬁrst one might exhibit unfavourable ﬂuctuations. For a detailed overview and an illustration of the setup, see Hirschi et. al. (2019) [67]. Finally, the data can be corrected for continuous pH drift by making the following approximation. • A continuous piecewise linear function is constructed from the starting points of each illumination cycle and subtracted from the pH curve. This will set the starting points of each cycle to 0, thus converting the scale to relative pH change and removing any drift occurring during the recording. For a more detailed description of this process consult Harder et. al. (2016) [68]. MethodsMethods Protoc.Protoc. 20202020,, 33,, xx FORFOR PEERPEER REVIEWREVIEW 14 14 of of 21 21

thethe firstfirst oneone mightmight exhibitexhibit unfavourableunfavourable fluctuatiofluctuations.ns. ForFor aa detaileddetailed overviewoverview andand anan illustrationillustration ofof thethe setup,setup, seesee HirschiHirschi et.et. al.al. (2019)(2019) [67].[67]. •• Finally,Finally, thethe datadata cancan bebe correctedcorrected forfor contcontinuousinuous pHpH driftdrift byby makingmaking thethe followingfollowing approximation.approximation. AA continuouscontinuous piecewisepiecewise linearlinear functionfunction isis constructedconstructed fromfrom thethe startingstarting pointspoints ofof eacheach illuminationillumination cyclecycle andand subtractedsubtracted fromfrom thethe pHpH curve.curve. ThisThis willwill setset thethe startingstarting pointspoints ofof eacheach cyclecycle toto 0,0, thusthus convertingconverting thethe scalescale toto rerelativelative pHpH changechange andand removingremoving anyany driftdrift occurringoccurring Methodsduringduring Protoc. the2020the recording.,recording.3, 51 ForFor aa moremore detaileddetailed descriptidescriptionon ofof thisthis processprocess consultconsult HarderHarder et.et. al.al. (2016)15(2016) of 22 [68].[68].

FigureFigure 4.4. PhotoactivityPhotoactivity of of BR BR versionsversions reconstitutedreconstituted intointointo liposomes.liposomes.liposomes. Drift-corrected Drift-corrected photoactivity photoactivity

measurementsmeasurements of of measuringmeasuring solutionsolution (Background)(Background)(Background) ororor BR-wt,BR-wt,BR-wt, BR-C-HisBR-C-HisBR-C-His101010 andandand BR-D98N-C-HisBR-D98N-C-HisBR-D98N-C-His101010 proteoliposomes.proteoliposomes. YellowYellow Yellow andand graygray shading shading indicate indicate 15 15 min min light light and and darkdark cycles.cycles. TheThe pH pH of of the the

extracellularextracellular solutionsolution ofof proteoliposomesproteoliposomes containingcontcontainingaining functionalfunctional BR BRBR versionsversions (wt(wt andand C-HisC-His101010))) increases increases uponupon illumination,illumination,illumination, demonstrating demonstratingdemonstrating the thethe proton protonproton pumping pumpinpumpin activitygg activityactivity and thus andand functional thusthus functionalfunctional insertion. insertion.insertion. In contrast InIn

contrastBR-D96N-C-Hiscontrast BR-D96N-C-HisBR-D96N-C-His10 proteoliposomes1010 proteoliposomesproteoliposomes exhibit only exhibitexhibit background onlyonly levelbackgroundbackground pH change levellevel upon pHpH illumination changechange uponupon since illuminationD96Nillumination is a BR sincesince version D96ND96N with isis defectiveaa BRBR versionversion proton withwith pumping defedefectivective activity. protonproton Allpumpingpumping proteoliposomes activity.activity. AllAll were proteoliposomesproteoliposomes reconstituted werewithwere the reconstitutedreconstituted same amount withwith of thethe protein; samesame thus, amountamount the of increasedof proteiprotein;n; pH thus,thus, change thethe increasedincreased of the His-tagged pHpH changechange version ofof thethe is His-taggedHis-tagged either due versiontoversion higher isis protein eithereither duedue activity toto higherhigher or more proteinprotein eﬃcient acactivitytivity reconstitution. oror moremore effiefficientcient reconstitution.reconstitution.

3.18.3.18. Structural Structural Characterization Characterization of of Purple Purple Membranes Membranes by by Negative Negative Stain Stain Transmission Transmission ElectronElectron Microscopy (1 Day) MicroscopyMicroscopy (1(1 Day)Day) Prepare 0.75% (w/v) uranyl formate solution and filter it with a 0.2 µm syringe filter before use to •• PreparePrepare 0.75%0.75% (w/v)(w/v) uranyluranyl formateformate solutionsolution andand fifilterlter itit withwith aa 0.20.2 µmµm syringesyringe filterfilter beforebefore useuse remove any precipitates. toto removeremove anyany precipitates.precipitates. Carbon-coated copper grids are placed with the coated (shiny) side facing up on a •• Carbon-coatedCarbon-coated coppercopper gridsgrids areare placedplaced withwith thethe coatedcoated (shiny)(shiny) sideside facingfacing upup onon aa Parafilm-Parafilm- Parafilm-wrapped microscope slide. Use precision forceps while handling microscope grids and wrappedwrapped microscopemicroscope slide.slide. UseUse precisionprecision forcepsforceps whilewhile handlinghandling microscopemicroscope gridsgrids andand onlyonly only hold them on the edges to avoid damaging the center. Grids are then glow discharged for 10 holdhold themthem onon thethe edgesedges toto avoidavoid damagingdamaging thethe cecenter.nter. GridsGrids areare thenthen glowglow dischargeddischarged forfor 1010 ss s under vacuum. underunder vacuum.vacuum. CRITICALCRITICAL STEP STEP GlowGlow discharging discharging electron electron microscopy microscopy grids grids is is essential essential for for the the efficient eefficientfficient adsorptionadsorption ofof hydrophilic hydrophilic samples samples such such as as prot proteoliposomesproteoliposomeseoliposomes to to the the hydrophobic hydrophobic grids. grids. However,However, However, excessiveexcessive glowglow discharging discharging shouldshould bebe avoided avoided as as it it can can damage damage the the extremely extremely thin thin carbon carbon layer. layer. •• AA totaltotal of of 5 5 µLµµLL of of purple purple membrane membrane at at a a concentration concentration of of about about 1 1 mg/mL mgmg/mL/mL is is adsorbed adsorbed onto onto a a glow glow • dischargeddischarged gridgrid forfor 11 min.min. •• TheThe liquidliquid isis removed removed byby gentlygently touchingtouching thethe sideside ofof thethe grid grid with with blotting blotting paper. paper. Then, Then, the the grid grid • isis immediately immediately washed washedwashed three threethree times timestimes by byby placing placingplacing a a 5 5 µLµµLL drop drop of of ultrapure ultrapure water water on on the the grid grid and and removingremoving itit usingusing thethe blottingblotting paper.paper. •• AA total total of of 5 5 µµLµLL ofof uranyluranyl formateformate solutionsolution isis placedplplacedaced on on thethe gridgrid andand immediatelyimmediately blotted. blotted. Then, Then, • anotheranother drop drop is is added added and and incubated incubated for for 10 10 s s before before drying drying thethe gridgrid againagain withwith blottingblotting paper.paper. Finally,Finally, letlet thethethe gridsgridsgrids airairair drydrydry forforfor aaa fewfewfew minutesminutesminutes before beforebefore placing placingplacing them themthem in inin a aa grid gridgrid storage storagestorage box. box.box. The general procedure of operating a transmission electron microscope can be obtained from the • operating manual. The size and morphology of the purple membrane patches (see Figure5) can be assessed at low magnification (e.g., 5–10,000 ). The structural integrity and organization of × the BR crystals can be assessed by imaging the patches at higher magnification (e.g., 68,000 ) × and observing the power spectrum via the Fourier transformation of corresponding images (see Figure5 insets). Methods Protoc. 2020, 3, x FOR PEER REVIEW 15 of 21

• The general procedure of operating a transmission electron microscope can be obtained from the operating manual. The size and morphology of the purple membrane patches (see Figure 5) can be assessed at low magnification (e.g., 5–10,000×). The structural integrity and organization of the BR crystals can be assessed by imaging the patches at higher magnification (e.g., 68,000×) Methods Protoc.and observing2020, 3, 51 the power spectrum via the Fourier transformation of corresponding images (see16 of 22 Figure 5 insets).

FigureFigure 5. Structural5. Structural characterization characterization ofof purplepurple membrane membraness by by negative negative stain stain transmission transmission electron electron microscopy.microscopy. Electron Electron micrographs micrographs of of negativelynegatively st stainedained purple purple membrane membrane patches patches containing containing BR-wt BR-wt (a), BR-C-His10 (b) and BR-D96N-C-His10 (c). Insets show the power spectra of selected areas at higher (a), BR-C-His10 (b) and BR-D96N-C-His10 (c). Insets show the power spectra of selected areas at higher magnification and demonstrate the crystallinity of the membrane patches. Scale bars represent 500 Å magnification and demonstrate the crystallinity of the membrane patches. Scale bars represent 500 Å −1 and 50 1Å in the insets. and 50 Å− in the insets. 4. Expected Results 4. Expected Results We have presented a detailed protocol on the production of an engineered BR version having a C-terminalWe have presentedHis10-tag (BR-C-His a detailed10). protocolFurthermore, on the we production have described of an the engineered isolation procedure, BR version and having a C-terminalfunctional and His 10structural-tag (BR-C-His analysis 10of). the Furthermore, resulting PMs wecontaining have describedHis-tagged theBR. isolationImportantly, procedure, this andprotocol functional opens and the structural possibility analysis to scientists of the resultingto tailor own PMs versions containing of BR His-tagged for specific BR. applications Importantly, in this protocolbasic opensresearch the and possibility applied sciences. to scientists to tailor own versions of BR for specific applications in basic researchWe and first applied introduced sciences. a His10-tag at the C-terminus of the bop gene in the pUC18 vector and subsequently cloned the bop-C-His10 gene into the pHS blue vector. H. salinarum L33 cells were then We first introduced a His10-tag at the C-terminus of the bop gene in the pUC18 vector and transformed with the resulting construct, and the PMs of several clones were isolated and analysed subsequently cloned the bop-C-His10 gene into the pHS blue vector. H. salinarum L33 cells were then by SDS-PAGE (Figure 3). The BR-deficient H. salinarum L33 [53,54] strain has the tendency to produce transformed with the resulting construct, and the PMs of several clones were isolated and analysed by revertants via homologous recombination between the transformed bop gene copy and the native bop SDS-PAGE (Figure3). The BR-deficient H. salinarum L33 [53,54] strain has the tendency to produce gene [46,69], which is here partially disrupted by an insertion element [54]. This might result in purple revertants via homologous recombination between the transformed bop gene copy and the native membranes containing mixtures of BR-wt and the desired engineered version, e.g., BR-C-His10 bop (Figuregene [46 3).,69 To], avoid which this, is hererounds partially of clone disrupted optimization by as an described insertion in element the protocol [54]. are This necessary might resultand in purplewere membranes applied to obtain containing a clone mixtures producing of BR-wt maximal and amounts the desired of His-tagged engineered BR version, (Figure e.g.,3). After BR-C-His the 10 (Figuresuccessful3). To avoididentification this, rounds of a suitable of clone clone, optimization larger cultures as described were grown in the and protocol the isolated are necessary PMs were and werebiochemically, applied to obtain functionally a clone and producing structurally maximal analysed. amounts of His-tagged BR (Figure3). After the successfulBR-C-His identification10 and BR-wt of a suitableas control clone, were largerreconstitute culturesd using were DOPC grown lipids and and the isolatedtheir functions PMs were biochemically,studied using functionally a photoactivity and structurallyassay. Upon analysed.illumination, BRs incorporated in the proteoliposomes pump protons into the lumen, which can be detected in the surrounding medium using a micro pH- BR-C-His10 and BR-wt as control were reconstituted using DOPC lipids and their functions studiedelectrode using (Figure a photoactivity 4). Proteolip assay.osomes Upon containing illumination, BR-C-His BRs incorporated10 or BR-wt produced in the proteoliposomes a pH-gradient of pump comparable amplitude upon illumination, which decays when the illumination is stopped. This protons into the lumen, which can be detected in the surrounding medium using a micro pH-electrode shows that the C-terminal His10-tag is not interfering with the proton pumping function of BR. (Figure4). Proteoliposomes containing BR-C-His 10 or BR-wt produced a pH-gradient of comparable Transmission electron microscopy analysis of PMs containing BR-C-His10 (Figure 5b) shows amplitudecomparable upon sizes illumination, and shapes whichof membrane decays patches when as the for illumination PMs containing is stopped. BR-wt (Figure This shows5a). Power that the C-terminalspectra calculated His10-tag from is not electron interfering micrographs with the indica protonted pumping that His-tagged function PM of patches BR. Transmission possess the same electron microscopyhighly ordered analysis hexagonal of PMs 2D containing crystal structure BR-C-His (Figur10 (Figuree 5b, inset)5b)shows as PMs comparable containing BR-wt sizes (Figure and shapes 5a, of membraneinset). This patches demonstrates as for PMs that containing the introduction BR-wt of (Figure a C-terminal5a). Power His10-tag spectra is not calculated interfering from with electron the micrographsformation of indicated the typical that PM His-tagged structure. PM patches possess the same highly ordered hexagonal 2D crystal structure (Figure5b, inset) as PMs containing BR-wt (Figure5a, inset). This demonstrates that the introduction of a C-terminal His10-tag is not interfering with the formation of the typical PM structure. Building on BR-C-His10, a functional mutant of BR that has a significantly prolonged M-state, i.e., ~1000-fold longer than that of BR-wt, was created by introducing a point mutation into the His-tagged construct (BR-D96N-C-His10)[37,70]. For such mutants, it is important to introduce the mutation into a BR version, which has a different molecular mass than the wild-type form, e.g., BR-C-His10, in order to be able to differentiate between mutant version and revertant wild-type BR by SDS-PAGE. The point mutation leading to a version of BR in which the aspartate at position 96 is exchanged for an Methods Protoc. 2020, 3, x FOR PEER REVIEW 16 of 21

Building on BR-C-His10, a functional mutant of BR that has a significantly prolonged M-state, i.e., ~1000-fold longer than that of BR-wt, was created by introducing a point mutation into the His- tagged construct (BR-D96N-C-His10) [37,70]. For such mutants, it is important to introduce the mutation into a BR version, which has a different molecular mass than the wild-type form, e.g., BR-

C-HisMethods10 Protoc., in order2020, 3to, 51 be able to differentiate between mutant version and revertant wild-type BR17 of by 22 SDS-PAGE. The point mutation leading to a version of BR in which the aspartate at position 96 is exchanged for an asparagine, was introduced into the bop-C-His10 gene in the pUC18 vector followed byasparagine, cloning into was the introduced pHS blue into vector. the bop-C-His Next, H.10 salinarumgene in the L33 pUC18 was transformed vector followed with by the cloning resulting into constructthe pHS blue (pHS vector. blue BR-D96N-C-His Next, H. salinarum10) andL33 the was PMs transformed of several withclones the were resulting isolated. construct Already (pHS after blue the firstBR-D96N-C-His round of testing,10) and a the clone PMs only of several expressing clones BR-D96N-C-His were isolated. Already10, and no after BR-wt, the first could round be obtained. of testing, PMsa clone of onlya larger expressing culture BR-D96N-C-Hisof the selected clone10, and were no BR-wt, isolated, could and be the obtained. purity and PMs integrity of a larger assessed culture by of SDS-PAGEthe selected (Figure clone were 6a) and isolated, Western and blot the purityanalysis and (F integrityigure 6b). assessed The former by SDS-PAGE showed only (Figure one 6banda) and at Westernthe expected blot analysis size and (Figure no BR-wt6b). The band, former while showed the later only onetechnique band at confirmed the expected the size presence and no BR-wtof the introducedband, while His-tag. the later A technique less prominent confirmed band thearound presence 50 kDa, of theindicated introduced the presence His-tag. of A a lessminor prominent fraction ofband SDS-resistant around 50 BR-D96N-C-His kDa, indicated the10 dimers presence [38,39]. of a This minor purple fraction membrane of SDS-resistant preparation BR-D96N-C-His yielded 2.5 mg10 ofdimers BR-D96N-C-His [38,39]. This10 (for purple BR quantifications membrane preparation refer to 5.2 yielded Equipment 2.5 mg Setup) of BR-D96N-C-His per liter of cell10 culture.(for BR quantificationsWhile yields can refer differ to Section significantly 5.2) per between liter of cell differen culture.t constructs, While yields yields can di offf er~5 significantly mg per liter between of cell culturedifferent for constructs, wild-type yields BR from of ~5 H. mg salinarum per liter of S9 cell and culture ~1–3 formg wild-type per liter BRof fromcell cultureH. salinarum for mutantsS9 and ~1–3expressed mg per in literH. salinarum of cell culture L33 can for be mutants expected. expressed in H. salinarum L33 can be expected.

Figure 6. 6. AnalysisAnalysis of BR-D96N-C-His of BR-D96N-C-His10 by10 SDS-PAGEby SDS-PAGE and Western and Western blotting. blotting. (a) Coomassie (a) Coomassie Brilliant blue-stainedBrilliant blue-stained 13.5% SDS/polyacrylamide 13.5% SDS/polyacrylamide gel of isolated gel of isolated purple purple membranes membranes from fromH. salinarumH. salinarum L33

transformedL33 transformed with BR-D96N-C-His with BR-D96N-C-His10 and 10BR-wtand (isolated BR-wt (isolated from H. salinarum from H. salinarumS9) as a control.S9) as BR-D96N- a control. C-HisBR-D96N-C-His10 migrates10 slowermigrates compared slower to compared BR-wt, because to BR-wt, of the because additional of the His additional10-tag. InHis each10 -tag.lane, Inpurple each membraneslane, purple corresponding membranes corresponding to 10 µg of bacteriorhodopsin to 10 µg of bacteriorhodopsin were loaded. were(b) The loaded. Western (b) Theblot Westernanalysis

ofblot BR-wt analysis and of BR-wtBR-D96N-C-His and BR-D96N-C-His10 performed10 performed under similar under conditions similar conditions as in (a as). inThe (a ).primary The primary and secondaryand secondary antibodies antibodies used usedwerewere mouse mouse monoclonal monoclonal anti-penta-His anti-penta-His antibo antibodydy (dilution (dilution 1:3000) 1:3000) and horseradishand horseradish peroxidase-conjugated peroxidase-conjugated goat anti-mouse goat anti-mouse antibody antibody (dilution (dilution 1:3000), 1:3000), respectively. respectively. BR-wt showedBR-wt showed no band, no due band, to the due absence to the of absence a His10-tag. of a BR-D96N-C-His His10-tag. BR-D96N-C-His10 reacted with10 thereacted anti-penta-His with the antibody,anti-penta-His confirming antibody, the conﬁrming presence theof the presence introduced of the introducedHis10-tag. A His weaker10-tag. Aband weaker around band 50 around kDa, indicates50 kDa, indicates the presence the presence of SDS-resistant of SDS-resistant BR-D96N-C-His BR-D96N-C-His10 dimers10 [38,39].dimers [38,39].

Analogous to to BR-wt BR-wt and and BR-C-His BR-C-His10, BR-D96N-C-His10, BR-D96N-C-His10 was10 reconstitutedwas reconstituted in DOPC in DOPClipids and lipids its functionaland its functional properties properties were analysed were using analysed the phot usingoactivity the photoactivity assay described assay previously described (Figure previously 4). In (Figurecontrast4 ).to In BR-C-His contrast to10, BR-C-His BR-D96N-C-His10, BR-D96N-C-His10 did not 10producedid not a produce pH change a pH changeupon illumination. upon illumination. This Thisconfirms conﬁrms the expected the expected slowing slowing of the of theproton proton pumping pumping activity activity by by the the introduction introduction of of the the D96N mutation. To To test, if the isolated mutant PMs form the same distinct patches and have the same 2D crystal structure, they were struct structurallyurally analysed by negative stain transmission electron electron microscopy (Figure5 5c).c). TheThe electronelectron micrographsmicrographsof of thethe PMsPMs containingcontaining BR-D96N-C-HisBR-D96N-C-His1010 showedshowed similar similar shapes and sizes of patches as the ones for PMs containing BR-wt (Figure5 5a)a) andand BR-C-HisBR-C-His10 (Figure(Figure 55b).b). Analysis of calculated power spectra conﬁrmed that the PMs retain their typical hexagonal 2D crystal

lattice (Figure5c, inset). The obtained results from BR versions show that the presented protocol is applicable for the production of BR variants containing a purification tag or functional mutation. Importantly, the engineered modifications had no effect on the 2D assembly into PMs preserving this unique and highly valuable feature. Methods Protoc. 2020, 3, 51 18 of 22

5. Setup

5.1. Reagent Setup TAE buffer: Mix 40 mM Tris base, 20 mM acetic acid and 1 mM EDTA. LB medium: Dissolve 25 g/L LB Broth powder in deionized water and autoclave. LB-agar plates: Dissolve 25 g/L LB Broth powder in deionized water. Add 15 g/L Bacto™ Agar and autoclave. L37 medium: Mix 10.2 mM trisodium citrate dihydrate, 4.3 M NaCl, 80 mM MgSO4, 27 mM KCl, and 10 g/L bacteriological peptone in deionized water. Adjust with NaOH to pH 7 at room temperature and sterilize for 2 h at 90 ◦C in a dry sterilizer. Novobiocin stock solution: Dissolve 1 mg/mL novobiocin sodium salt in Milli-Q ultrapure water. Sterilize by filtration. L37-agar plates: Mix 10.2 mM trisodium citrate dihydrate, 4.3 M NaCl, 80 mM MgSO4, 27 mM KCl, and 10 g/L bacteriological peptone in deionized water. Adjust with NaOH to pH 7 at room temperature. Add 15 g/L Bacto™ Agar and sterilize for 2 h at 90 ◦C in a dry sterilizer. Spheroplast buffer (SPH): Mix 50 mM Tris-HCl (from 1 M stock solution at pH 8.75), 2 M NaCl, 27 mM KCl, and 150 g/L sucrose in Milli-Q ultrapure water. Sterilize by filtration. EDTA solution: Dissolve 500 mM EDTA in SPH. PEG solution: Mix 3 parts of PEG600 with 2 parts of SPH. Spheroplast dilution solution: Mix 10.2 mM trisodium citrate dihydrate, 50 mM Tris-HCl (from 2 M stock solution adjusted to pH 7.4), 4.3 M NaCl, 80 mM MgSO4, 27 mM KCl, 1.55 mM CaCl2, and 150 g/L sucrose in Milli-Q ultrapure water. Sterilize by filtration. Regeneration medium: Dissolve 150 g/L sucrose in L37 medium. Sterilize by filtration. 50% (v/v) glycerol solution: Mix glycerol and Milli-Q ultrapure water 1:1 and autoclave. Basal salt: Mix 4.3 M NaCl, 80 mM MgSO4, and 27 mM KCl in deionized water and autoclave. DNAse solution: Prepare a 2.5 mg/mL solution from the lyophilized DNAse using Milli-Q ultrapure water. Tris-buffered saline (TBS): Mix 50 mM Tris and 150 mM NaCl in Milli-Q ultrapure water and adjust the pH to pH 8. Hydration buffer: Mix 0.76 g/L KH2PO4, 2.51 g/LK2HPO4 and 100 mM KCl in Milli-Q ultrapure water and confirm the pH. If it is not 7.2, adjust accordingly. Dialysis buffer: Mix 20 mM Tris and 150 mM NaCl in cold Milli-Q ultrapure water. Adjust the pH with HCl to 8 at 4 ◦C and fill up to 2 L with Milli-Q ultrapure water. Measuring solution: Adjust a solution of 150 mM NaCl to pH 7.4 with NaOH and always check the pH immediately before use. Optionally, degas the solution with nitrogen gas to increase the pH stability.

5.2. Equipment Setup L37-Agar plates: Due to the high sodium chloride concentration of L37-agar plates and the long incubation time (up to several weeks), it is necessary to seal the plates airtight, to prevent evaporation and salt crystal formation. This can be achieved by packing the plates into sealed plastic bags or by sealing the plates with several rounds of a standard plastic adhesive tape prior to incubation. Chromophore-based protein quantiﬁcation: To quantify the concentration of bacteriorhodopsin in a purple membrane suspension, the absorption at 568 nm is measured using a spectrophotometer. We use the UV-Vis program of a Thermo Fisher NanoDrop 1000 with measurements at 280 nm and 568 nm. High-quality purple membranes show a A280/A568 ratio of 2 [1] and lower. A higher ratio indicates the presence of proteins other than bacteriorhodopsin. The concentration of bacteriorhodopsin in suspensions of purple membranes can be estimated by using a molar extinction coeﬃcient of 63,000 L mol 1 cm 1 at 568 nm [6]. · − · − Methods Protoc. 2020, 3, 51 19 of 22

Supplementary Materials: The following are available online at http://www.mdpi.com/2409-9279/3/3/51/s1, Material S1: Nucleotide sequence of pHS blue, Figure S1: Vector map of pHS blue. Author Contributions: Conceptualization, M.S., S.H., D.H., R.S. and D.F.; methodology, M.S., S.H., Z.U., D.H., R.S. and D.F.; formal analysis, M.S., S.H., Z.U., D.H., R.S. and D.F.; investigation, M.S., S.H., D.H. and Z.U.; resources, Z.U., R.S. and D.F.; writing—original draft preparation, M.S., S.H. and D.F.; writing—review and editing, M.S., S.H., R.S. and D.F.; visualization, M.S. and S.H.; supervision, D.F.; funding acquisition, R.S. and D.F. All authors have read and agree to the published version of the manuscript. Funding: This work was supported by the University of Bern, the National Centre of Competence in Research (NCCR) Molecular Systems Engineering and the Swiss National Science Foundation (SNSF), as well as by the German Research Foundation via the SFB-1078 project B4 (to R.S.). Acknowledgments: The authors thank Ulf Eidhoff for providing the pHS blue vector. Electron microscopy experiments were performed on equipment supported by the Microscopy Imaging Center (MIC) of the University of Bern, Switzerland. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

References

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