© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. measures to ensure reproducible material production. microscopy ( characterizing hierarchical phage nanostructures using optical microscopy, atomic-force microscopy ( genetic engineering of phage, and purification, liter-scale amplification, assembly,self-templating and suggest approaches for regenerating materials, biosensors, and energy-generating materials. Here, we describe a comprehensive protocol to perform genetic modification of functional peptides of resultsbacteriophage in structures that can be used as soft and hard tissue- its liquid crystalline structure for the creation of diverse hierarchically organized structures. We have also demonstrated that based biomimetic process, called assembly’.‘self-templating We used as bacteriophage a nanofiber model system to exploit functions are still very limited, and scalable synthesis is far from being realized. assembly processes have been developed to exploit biological molecules for functional materials, the resulting nanostructures and and fornanotechnology production of electronic devices, energy generators, biosensors, and bionanomedicines. L Published online 31 August 2017; and Development Center, Vicksburg, MS, USA. Center, Vicksburg, MS, USA. Laboratory, Berkeley, CA, USA. 1 Seung-Wuk Lee oaig arcs n ooe mmain kn, n helicoi and structures bone skins, arranged dally mammalian colored on matrices rotating cholesterically eyes, the in tissues corneal arranged orthogonally structures struc tures that combine to arrange macroscopic organized hierarchical self-assembled micrometer-level or nanometer- the lize stabi states These factors. thermodynamic and kinetic controls of secretion the blocks helical building spontaneously on-and-off periodic The conditions. given the in structures stable most the assemble blocks building helical the terminates, secretion When direction. flow the to parallel fiber the of orientation the makes flow and forces induces Secretion materials. ordered highly form to states equilibrium from far favor momentarily that ronments mol helical ecules incoming of orientation the influence structures, preexisiting and curvatures, spaces, confined as such ronments, orientations molecular rotating or twisting, parallel, into self-organize nanofibers the bly,which in assem to lead self-templated microenvironments cellular within interactions molecular the of control of and nanofibers secretion helical the timely The structure. hierarchical a in helical nanofibers organizing in role critical a have factors environmental and kinetic process, this In process. morphogenesis the during celluloses and chitins, as collagens, such nanofibers helical self-assem of bly through nanostructures functional various creates processes characterization performance and design laborious, but rational, through generated often are particularly science, when these materials materials have nanometer-scale in dimensions. Materials challenge a is functions able desir and structures well-defined with materials new Designing I Ju Hun Lee hierarchically assembled bacteriophages Production of tunable nanomaterials using microscopic tools to investigate the the investigate to tools of number limited microscopic and blocks building biological basic the of Department of Bioengineering, University Bioengineering, of Department California,of Berkeley, Berkeley, CA, USA. NTRO arge-scale arge-scale fabrication of precisely defined nanostructures with tunable functions is critical to the exploitation of nanoscience 4, D 5 UCT . These self-assembly processes occur in controlled envi in occur controlled processes . self-assembly These 6– 9 I SE 1 . Examples of such hierarchical structures include include structures hierarchical such of Examples . ON – 3 M). M). We also discuss sources of common contamination, mistakes during the fabrication process, and quality-control , 7 , Christopher M Warner 1 – 3 5 College of Pharmacy,College of Ajou University, Suwon, Republic Korea.of 3 Tsinghua-Berkeley Shenzhen Institute, Berkeley, CA, USA. doi:10. 10–1 1038/nprot.2017.08 2 2, © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. . Because of the complexity complexity the of Because . 3 7 These These authors contributed equally to this work. Correspondence should be addressed to S.-W.L. ( . Furthermore, local envi local Furthermore, . n situ in 1 . By contrast, nature nature contrast, By . 4 , 7 morphogenesis morphogenesis , Hyo-Eon Jin 5 T he he protocol takes ~8–10 d to complete. ------1

, 2 , 5 2 , viral particles viral have been developed using self-assembly of genetically engineered nanomaterials functional various nature, in processes self-templating assembly mimicking by Recently, ideal. from far is level assembly molecular the on celluloses self-templating and chitins, collagens, the of processes of understanding our process, adsorbs onto the substrate and crystallizes out of the meniscus. meniscus. the of out crystallizes and substrate the onto adsorbs solution phage condensed the process, evaporation the during meniscus the from vertically pulled are substrates When meet. phases only smectic near the where meniscus air, liquid, and solid phase transition of the phage can induce nematic, cholesteric, and of the phage solution at evaporation the spontaneous meniscus, a self- ( the nature in mimic process to templating process deposition particle phage induced transitions phase their and phases) smectic and cholesteric, nematic, (i.e., structures as used been a to system model crystalline study liquid large at ( produced scales be can phages monodisperse identical cally coli Escherichia proteins coat the of modification genetic through tides ( ref. end left; either at located pIX and pIII proteins coat minor the of copies five additional an with symmetry helical fivefold a with proteins coat major pVIII the of copies 2,700 by covered is a a M13 with surface, shape fibrous which phage possesses helical and celluloses), chitins, (i.e., collagens, nanofibers helical natural ( diameter in nm 6.6 and length in nm ~880 virus bacterial filamentous a is phage M13 structures. crystalline liquid self-assembled its and (phage) bacteriophage M13 is of shape to diverse nanofiber the structures exploit helical Division of Biological Systems Biological of Division and LawrenceEngineering, Berkeley National 7 , Barnes Eftihia An ideal starting point for designing helical-nanofiber-based helical-nanofiber-based designing for point starting ideal An 4 Environmental Laboratory, U.S. Army ResearchEngineer and Development 6 Geotechnical Geotechnical and Laboratory,Structures U.S. Army ResearchEngineer Fig. Fig. 1 8 ). Phage can be easily engineered with functional pep functional with engineered ). be can easily Phage 15,20–2 1 , center). Because of its monodispersity, phage has has phage monodispersity, its of Because center). , R 13–1 ( ecently, we have established a bacteriophage- E. E. coli 4 5 Rcnl, e eeoe a aie meniscus- facile a developed we Recently, . . 6 , Aimee R Poda natureprotocols ) bacterial host cells, chemically and host cells, ) physi chemically bacterial

Fig. Fig. A FM), FM), and scanning electron Fig. Fig. 1 , right; ref. ref. right; , 4 , Edward J Perkins

1 | VOL.12 NO.9VOL.12 ; refs. refs. ; A [email protected] lthough lthough self- 16,1 protocol 1 3 7 ). Exploiting Exploiting ). ). Similar to to Similar ). | 2017 1 9 . Using Fig. Fig. |

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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. followed by self-assembly and characterization of hierarchical hierarchical nanostructures. phage of characterization purification, and and self-assembly by followed amplification liter-scale for phages, neered materials composite inorganic–organic generation and storage energy biosensors for used be can material phage self-templated phases) nanofilament helicoidal smectic and ribbon, helical cholesteric twist, orthogonal nematic (i.e., nature the in observed never are or as collagen- tissue well chitin-like structures) as that structures (i.e., structures existing naturally diverse create can we process, precise at phage sub locations deposit on solid the substrateselectively to the controlled of be can strate functionalization) surface hydrophobic hydrophilic (i.e., or wettability the addition, In surface. the onto force field adsorbed with the that and interacts substrate particles the tune can we speed, pulling the of control Through together. particles phage forces bringing tion, we can the tune electrostatic concentra the assembly.ionic By controlling the self-templating we concentration, can in engaged control the number particles of phage the by controlling example, For scale. a at large structures interac the nano hierarchical of and the self-templating at tion the meniscus modulate to chemistry) surface or speed factors kinetic (pulling and solution) of concentration concen ionic or solution tration (phage thermodynamic the control can We microenvironments. cellular in fibers helical natural of process deposition and secretion periodic the mimics phenomenon slip stick-and- recedes. so The oscillating front that the rapidly liquid ‘slips’ solution the and threshold, critical a surpasses energy the until interface at solid–liquid–vapor the energy surface dynamic the increase forces Evaporative position. ‘stick’ a in solution in phage maintain meniscus the at forces surface time, this During ref. from permission with adapted images right and manner. Left a self-templated in substrate the on film nanostructured phage the deposits which interface, air/liquid/solid the at induced is solution phage of the transition phase crystal a liquid suspensions, phage from pulled slowly is substrate As the films. nanostructured various into self-assembled be can suspension phage concentrated and pure highly resulting The (Right) precipitation. PEG through concentrated and purified be can solution phage The infection. bacterial through prepared be can phage engineered of genetically quantities Large (Center) engineering. genetic through (minor, orange) pIII and (minor, green), pIX (major, yellow), pVIII as such proteins capsid of various terminus N the near modified be can phage M13 axis. rotation screw a twofold and symmetry helical a fivefold with proteins capsid major pVIII ~2,700 by 1 Figure 2000 protocol Here, we describe a comprehensive protocol to generate engi generate to protocol comprehensive a describe we Here,

pIX | VOL.12 NO.9VOL.12 genom 6.6 nm

1 | 1 M1 Genetic engineering 3 Schematic illustration of phage-based nanomaterials development process. (Left) Helical nanofiber-shaped phage structure. Phage is covered covered is Phage structure. phage nanofiber-shaped Helical (Left) process. development nanomaterials of phage-based illustration Schematic pVIII 3 . After controlled modification of phage, the resulting resulting the phage, of modification controlled . After , Nature Publishing Group. Group. Publishing , Nature 3 (Steps 1–12) e pIII pVIII pIII | pIX 2017 M13 phag | 25,2

natureprotocols e 6

. Using the self-templating film growth 880 nm 28,2 9 , tissue regeneration , tissue © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 13,3 E. coli 2 . amplificatio Phage Liter-scale amplification n 30,3 (Steps 13–44) 14,15,2 1 , and , and 7 - - - - - ,

materials metallic and semiconductor nanocrystals useful for battery-electrodefilmscould furtherbefunctionalized through nucleation target of fabricated anionic be could films phage and self-supporting polyethylenimine) (linear acid)polymerpolyelectrolyte(polyacrylic multilayers, large-scale cationic by assisted phage, M13 of nanomaterials deposition layer-by-layer through phage-based Furthermore, into and nanoparticles organic inorganic organize could phage the nanomaterials, various with interaction enable to modification genetic After films. and fibers crystalline liquid self-standing form could phage M13 that Lee applications. materials functional various coworkersandBelcher limited technique’s the capability and for cantilevers large-scale delicate patterning and expensive hinder for widespread need usage. the patterning, biological and molecules of control into research new prompted has development this Although tool. patterning preciselycontrolledamolecular- scanning-probe as cantilevertip a of application the on based nanolithography dip-peninvented qimn, o arct mse mls Mri ad colleagues and Mirkin molds. master fabricate to equipment, lithography beam electron orphotolithography as suchfacilities, largepatterns. Thismethod, however, requires relatively high-cost in choice of materials but also compatibility in fabricating relatively soft substrates transferring by structures nanoscale molecules on desired and substrates, such microscale as planar, both curved, flexible, of and types various fabricate to stamp elastomeric an with technology Whiteside and collaborators have developed a microcontact writing, printingmicrocontact printing, and dip-pen nanolithography accomplished by several approaches,been has includinggeometries different ink-jetinto biomaterials printing, of Self-assembly laser assembly methods self- biomolecule reported previously with Comparison purification Phage 4 2 . Although this method was used to successfully fabricate 3 4 . Microcontact printing provides not only flexibility 2 2

developed M13 phage self-assemblyphageand developed M13 Surface tension Surface

(Steps 45–53) Self-assembly Friction Pulling t al. et 4 1 . The resulting The . 22,3 9 showed 33–3 22,4 3 0 8 5

. .

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ecules at the molecular level within a relatively short amount amount short time relatively of a within level molecular the at ecules mol target desired of display, recognition for the phage it allows through targets various for substrates of motifs peptide binding Because phage provides a rapid mining tool to identify the specific molecular recognition elements to use them for color sensors receptor-like incorporate further Wecan color. structural bril liant exhibiting nanostructure bundled biomimetic a create to ings lation for communication, camouflage, and response to surround animals can rapidly change their colors through modu structural light scatter coherently that to nanostructures bundled attributed colors brilliant collagens, exhibiting (i.e., celluloses) and biomaterials chitins, structural of examples many are ( applications sensing for matrices color tions applica and sensing film phage colored structural Biomimetic materials tissue-engineering or generator, energy biosensor, for used be can that properties, biological and electrical, optical, including properties, their tunable and phage the with nanomaterials on generates structures self-assembled resulting proteins exposed of control Genetic phage self-assembled materials Application of nanofilaments helicoidal smectic and ribbons, helical cholesteric twists, orthogonal as nematic such synthesized, ously as many new structures that are neither found in nature nor previ chiral that structures liquid are crystalline found in nature, as well interplay and control of these factors, we have created the many novel, Through twist. higher-order and flow, material orientation, forces at interfacial the liquid crystal meniscus, which determines the of competition the is second The meniscus. the at phases tal process self-templating is the local of chiral transition liquid crys the during factor The twists. and first critical helical organization long-range, ordered structures with multiple levels of hierarchical produces process single-step This concentration). ionic and tion chemistry) and thermodynamic conditions (i.e., phage concentra surface and speed pulling (i.e., kinetic by the process the trolling con meniscus, air/solid/liquid the at films organized archically hier into phage the deposit to forces meniscus Wethe exploited substrate using vertically a computer-controlled pulling machine. phage and purification, we prepared a phage solution and pulled a process self-assembly induced meniscus- a using scale large a at films organized hierarchically into transformed are that nanostructures crystalline liquid form to blocks building nanofiber helical as used are particles phage that sense the in process assembly nanofiber helical natural the assembly’,method, termed assembly ‘self-templating that mimics self- the novel a to developed have we Recently, level. molecular the whole-organism from ranging structures refined into a ing in self-organiz through processes manner precise highly fabrication and bottom-up sophisticated in used are blocks ing build These sugars. and lipids, nucleotides, acids, as such amino 2D films with little hierarchical tunability. structures were limited to directionally organized liquid resultingcrystalline the functions, desired with film 2D functional scalable chemicals through a phage-display process and self-assembled self-assembled and process phage-display a through chemicals Natural biomaterials are composed of basic building blocks blocks building basic of composed are biomaterials Natural 47,4 The self-assembled phage films can be used as structural structural as used be can films phage self-assembled The 8 4 . Genetically engineered M13 phages can self-assemble self-assemble can phages M13 engineered Genetically . 9 . Recently, we engineered the phage to bind desired desired bind to phage the engineered we Recently, . 13,14,26,3 1 3 . After large-scale production of of production large-scale . After © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 0 . Fig. Fig. 2 a ). In nature, there ). there In nature, 43–4 1 3 . 6 . These .These 14,1 5 ------. having an alpha-helical structure with a dipole moment directed directed moment a dipole with structure an alpha-helical having axis phage the to angle tilted 20° the with symmetry screw helical twofold and rotational fivefold has and pVIII protein capsid major the of ies electric properties piezo exhibit and chitin cellulose, collagen, crystals, and protein as such DNA, acid amino energy. cal Many biomaterials, ordered into electri energy convert they mechanical meaning properties, piezoelectric exhibit can center inversion an without structure rials ( mate phage films can be used as piezoelectric-energy-generating generation piezoelectricity M13-phage-based designs sensor environmental and health various to applicable be can that targets desired to against respond engineered further be can film phage The TNT.colorimetric to resulting exposure upon changes color selective and sitive display, sen exhibited phage TNT-binding matrices the resulting engineered phages to bind a trinitrotoluene (TNT) through phage changes color characteristic exhibited matrices color phage the chemicals, desired of application Upon structures film nanostructured bundled into particles phage resulting the replicated in large quantities through bacterial amplification. amplification. bacterial through quantities large in replicated process tissue-growth the direct to structures mechanical and chemical, physical, well-defined of ing enables because phage the materials engineering construction tive nanofibrous basic building blocks for use in tissue-regenerat cells neighboring with stimuli physical and chemical various through behavior cellular nanofibrous structures in nature synergistically support and guide Hierarchical materials. tissue-regenerating in of issues the design vital most the are behavior cell regulate signaling and/or detect of to motifs engineering and arrangement structural of lation for ( tissue regeneration scaffold novel a offer can matrices self-assembled phage-based regeneration tissue M13-phage-based future. the in phage of properties piezoelectric the enhance can further film phage-nanostructured of self-assembly panel) LCD an (i.e., device a on microelectronic turn to mV,enough 400 of voltages produce and nA can 6 of devices currents piezoelectric phage-based fabricated of the level saturated a reaching d thickness, film increasing with increased coefficient the the film piezoelectric thickness: effective on In dependent was areas. response groove piezoelectric the observed the addition, of that than higher was areas ridge the of coefficient, piezoelectric effective the that revealed mapping ~1- with and position) orientation both films in (ordered phage structures resulting smectic the exhibited that verified characterization AFM materials these of relationships structure–property the gated investi and structures phage self-assembled of morphology the symmetry tric the piezoelec of the breaking of because stimulation mechanical upon piezoelectricity exhibit films monolayer phage assembled requirement material piezoelectric the satisfies structure from the N-terminus to the C-terminus direction. The M13 phage eff

≈ 3.9 pm/V for films thicker than ~100 nm. Furthermore, Furthermore, nm. ~100 than thicker films for pm/V 3.9 Fig. 2 1 b 4 . The resulting phage films exhibit a distinct color. distinct a exhibit films phage resulting The . ; ref. 26,5 1 8 . The pVIII protein consists of 50 amino acids acids amino 50 of consists protein pVIII The . 2 6 50–5 . To enhance piezoelectricity, we manipulated manipulated we .To piezoelectricity, enhance 6 ). Materials possessing a noncentrosymmetric µ m layer spacing. Piezoelectric-coefficient Piezoelectric-coefficient spacing. layer m 5 natureprotocols 58–6 . The M13 phage is covered with 2,700 cop Fig. 2 1 . attrac is the most M13 one phage of c ; refs. 6 2 . Furthermore, phages can be be can phages Furthermore, . 13,30,5 1 The genetically modified modified The genetically

4 | VOL.12 NO.9VOL.12 . For example, after we we after example, For . 1 5 . 7 ). A precise manipu 2 6 . The hierarchical hierarchical The . protocol Self-assembled Self-assembled | 2017 2 6 | . Self-

2001 d eff 2 6 ------, .

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. the self-assembled matrices of genetically engineered M13 phage engineered genetically of matrices the self-assembled that demonstrated have we Previously, behaviors. cell of nation exami quantitative allows surface phage nanometer-scaled the at motifs signaling displayed of density the tuning Furthermore, differentiation and proliferation and cell both of integration, enhancement material attachment, cell of improvement to nanometer-sized fibrous for structures cell that engagement leads ronment envi to a provide cell-conducive scaffold as a regeneration tissue act structures hierarchical nanofibrous phage-based Bioinspired from ref. the preosteoblast cells (MC-3T3 E1) grow to confluence (day 3) in the desired directions on the self-assembled RGD phage films. can self-assemble to form tissue-engineering matrices to guide the cellular growth process of the desired cells. Composite fluorescent imagetissue-engineering (right) showsmatrices. that The phage can be engineered to display high-density signaling peptides (1.5 × 10 generate electric potentials. The photograph depicts a phage-based piezoelectric device turning on an LCD device in response to acenters, finger thetap. M13 ( phage possesses intrinsic piezoelectric properties. Upon application of external forces, the phage and their selectively.self-assembled matrices ( can that can change colors upon binding of target molecules. Through color analysis and pattern recognition, we can detect various molecules sensitivelystructure and can be engineered to display receptors for desired chemicals. The resulting engineered phage can self-assemble to form colorimetricFigure 2 thin films 2002 scaffolds tissue-regenerating as used be can protocol

| VOL.12 NO.9VOL.12

1 | Engineered phage nanomaterial applications. ( 3 30,6 , Nature Publishing Group. Receptor-engineered b a c 1.5 ×10 b ) Phage-based piezoelectric-energy-generating matrices. Because of its noncentrosymmetric coat protein arrangement that has no inversion

RGD integrin-bindingpeptide 880 nm 2 pIII pI . These biomaterials provide large surface areas of of areas surface large provide biomaterials These . with receptors with cell-signalingmotifs pVIII Engineered M13phage M13 phage Engineered M13 phag X 13 RGDpeptidespercm | Engineered M13phag 2017 6.6 nm RG with morecharges pVIII e D |

natureprotocols pVIII arrangemen 2.7 nm 2 e © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. t 30,63–6 a phage matrixdirects ) Phage-based colorimetric sensor matrices. Through genetic engineering, the phage protein 5 Self-assembled . Phages were were Phages . cell growth phage nanofibers Self-assembled 30,6 phage piezoelectricmatrices Force applications Phage colorsensor 2 - - . Self-assembled

Au matrix arra phage structures and their alignment guided the cells to position to position cells the guided alignment their and structures phage hierarchical the that observed Wealso phage. wild-type of case the in observed that than cells of spread and migration greater a to led matrices phage engineered of specificity the that observed We differentiation. and proliferation cell) preosteoblast and cell progenitor (neural cell for scaffolds as serve can matrices phage manner compact and geneous homo a in protein capsid major pVIII the on alanine–valine)) the and neural-cell-stimulating peptide IKVAV (cell- (arginine–glycine–aspartate) (isoleucine–lysine–valine– RGD motifs peptide cell-signaling adhesive express to engineered genetically y e – 13 RGD peptides per cm Transitio 100 µ 3 m tissue-engineering matrix 0 Self-assembled phage . The resulting self-assembled self-assembled resulting The . n piezoelectric devices Phage-based sensor analysi c Phage color adapted with permission 2 ). The resulting phage s c ) Phage-based - © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. is a single-stranded DNA polynucleotide. However, in this pro this However,in polynucleotide. DNA single-stranded a is genome M13 the that note to important is It proteins. pVIII its on glutamates quadruple with phage M13 engineered an of case hybrids ecules, to or materials create piezoresponsive application-specific elements, recognition biomol with molecular phage engineering sites on the phage (i.e., at the pIII minor coat protein) to facilitate imple binding altering be in interested can are researchers Typically, that mented. modifications protein phage-coat of tions ( coats protein minor and/or on major its proteins, capsid) (minor pIII and pIX and protein capsid) (major pVIII the as such proteins, or tides engineering. genetic Phage fabricated. be can a materials phage processes, nanostructured of variety self-assembly phage and preparation, phage scale liter- the of the Through engineering, phage combination genetic design Experimental materials. sue-engineering tis hard and soft various design to used be can matrices phage tures found in nature that materials mimic dental enamel or tissues abalone shell struc strong and can tough form materials mechanically ized inorganic the on glutamates between phage and positive ions affinity the on based biominerals carbonate calcium and phosphate calcium nucleate can phages mineralization carbonate and as such in apatite tissue, hard role in biomineralizing critical well as materials assembled phages have also been used for hard tissue-engineering directions substrate-defined in themselves orient and T tocol, a double-stranded oligonucleotide from infected infected from oligonucleotide double-stranded a tocol, able able Biosensor Tissue engineering A Piezoelectricity pplication 1 1 22,4 |

Phage–protein engineering. 9 . In the following protocol, we describe the general general the describe we protocol, following the In . Fig. Fig. 1 3 . In nature, the glutamate-rich proteins have a have proteins glutamate-rich the . nature, In 1 ). Thus, there are many potential combina potential many are there Thus, ). 13,3 13,3 2 . Therefore, the self-assembled resulting 42,6 2 . The resulting phage with biomineral M13 phage can display desired pep desired display can phage M13 6 . Four glutamate-engineered (4E-) (4E-) glutamate-engineered .Four pVIII pVIII + pIII pVIII pVIII + pIII pVIII P hage engineering © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. 13,6 E. coli E. 2 . Self- . RGD RGD (pVIII), HPQ (pIII) E, EE, EEE, EEEE HPQ WHW HPQ (pVIII), (GPP) RGD (pVIII), HPQ (pIII) YGFGG PDPLEPRREVCE DGEA IKVAV RGD P E, E, EE, EEE, EEEE is is eptide ------

ples in in ples and exam pVIII. Weand/or pIII applications have specific listed to modification protein-coat expand can we application, phage desired the on Depending sequencing. DNA using confirmed is cloning method. The genome of the genetically engineered phages PCR inverse an using protein, coat major the of terminus N the at acids amino sixth and first the between desired inserted are the peptides proteins, surface the engineer genetically To used. the formation of phage films exhibiting liquid crystalline phases phases crystalline liquid exhibiting films phage of formation the to leading air, between (meniscus), and solid liquid, the interface at enhanced is rate pulled evaporation the are suspensions, phage the from substrates the When speeds. controlled precisely suspensions at phage from substrates solid pulls vertically which device, by a films using computer-controlled assem into large-area bled are Phages organization. hierarchical of levels multiple cess organizes phage particles into supramolecular structures with nanostructures various into phages engineered genetically of self-assembly the on based is protocol process. self-assembly Phage films. phage self-assembled create to tion solu stock phage engineered of preparation the regarding dures proce detailed describe we protocol, this In process. deposition the during nanostructures phage self-assembled of the production to vital also is preaggregation without pure solution A phage steps. M13 purification and amplification the during gates) aggre PEG and debris cell (e.g., biomaterials unwanted include might phages of amplification liter-scaled In general, phages. cal identi of a pure of solution highly is films phage the preparation cells host bacterial of ( infection through prepared are phages preparation. phage Liter-scale Fig. 3 ) Table . A critical factor in the fabrication of highly functional functional highly of fabrication the in factor critical A . 7 (pIII) 1 . natureprotocols The fabrication design in this this in design fabrication The Large quantities of identical identical of quantities Large

13,57,62,63,65,68,6 1 3 . The self-assembling pro self-assembling The .

14,15,72–7 R | VOL.12 NO.9VOL.12 eferences

26,5

7 1 7 7 6 7 7 6 3 6 5 5 1 3 0 0 4 0 6 protocol 4 | 2017 9 |

2003 ------

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. • • • • • • • • • • • • • • • • • • REAGENTS M with in various twisted orientations a hierarchical manner similar nanostructures factors, we substrates) crystalline have liquid tunable created the of chemistry surface and speed (pulling kinetic and solutions) the of concentration ionic and phage the of tion substrate (concentra the by By thermodynamic self-templating. manipulating onto particles phage of deposition and mulation accu local the to due phases) smectic and cholesteric, (nematic, 2004 • protocol Tetracycline (Sigma-Aldrich, cat. no. 87128) cat. no. D4551-500ML) DMF ( Magnesium chloride (Fisher Scientific, cat. no. BP214-500) Ethanol (Koptec, cat. no. V1016) Tris base (J.T. Baker, cat. no. 4109-02) chlorideSodium (Macron Chemical, cat. no. 7581-06) PEG 8000(Fisher Scientific, cat. no. BP233-1) Miller LBbroth (EMDChemicals, cat. no. 1.10285.0500) cat. no. 200228) XL1-Blue electroporation-competent cells (Agilent, E. coli QIAprep SpinMiniprep Kit (Qiagen, cat. no. 27104) QIAquick kit(Qiagen, PCRpurification cat. no. 28104) QIAquick Kit gelextraction (Qiagen, cat. no. 28704) T4 DNA (New ligase BioLabs, England cat. no. M0202S) PstI (New BioLabs, England cat. no. R0140S) cat. no. M0530S) DNA (NewPhusion high-fidelity polymerase BioLabs, England cat. no. 200523) QuikChange IIsite-directed mutagenesis kit(Agilent, O M13KE phage (New BioLabs, England cat. no. N0316S) ribbon structures canbeseenin and current are 10kVand 4–10nA,respectively. Examples of characterization results for right- and left-handed cholesteric helical phage fiberstructures. The phage structures do not need tohaveconductive coating for SEMinvestigation. The recommended voltage Characterize the long-range-ordered hierarchical phage structures using SEMtomonitor the uniformity and morphologies of individual S and smectic helicoidal nanofilaments canbeseenin could result inspurious image defects. Examples of characterization results for nematic stripedpatterns, nematic orthogonal twists, be operated insoft-tapping mode withAFMtipshaving aforce constant of 5–10N/mtoavoid undesirable phage–tip interactions that phage orientation and their local-ordered structures atarange of uptotens of micrometers. It isrecommended thatthe instrument Perform surface probe microscopy imaging tovisualize the nanoscopic structural morphologies of the resulting films to measure the A very usefulfor investigating nano/micro-fibrous-structure-dependent color generation. For evaluating the light-scattering properties of the phage film,a dark-field optical microscope canbeused. This experiment was with apolarizedoptical microscopy image (cross-polar) thatdetermined the directional order of phage structures onthe film. In addition, asshown in structures innematic stripe, nematic orthogonal twist,and cholesteric helical ribbonstructures canbeobserved( to which the filmisbehaving like aliquid crystal,apolarizedoptical microscope canbeused. For example, the groove and ridge Use optical microscopy methods toevaluate long-range-ordered microstructures inthe resulting films. To characterize the extent O take into account when analyzing phage films using the different approaches. The produced films canbecharacterized using optical microscopy, AFM,and SEM.Thisbox describes the different considerations to ATER Box 1 ligonucleotide (Integrated primers DNA Technologies, custom order) canning electron microscope tomic force microscope ptical microscope

| VOL.12 NO.9VOL.12 (strain: XL-1 (strain: Blue; Stratagene, cat. no. 200236) N I ALS , N -dimethylformamide; Sigma-Aldrich, | Filmcharacterization | 2017 |

natureprotocols Figure 6 Fig d , the polarizedoptical responses insmectic helicoidal nanofilament structures were observed © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. ure 6 c

.

● Fig

T ure IMI 6a , N b - - and G : 2–5h d • • • • • • • • • • EQUIPMENT • • • • • • • • ( before have that observed never been hierarchical structures as well as structures), bundled collagen teeth, and bone twisted helicoidally (i.e., structures hierarchical natural to dark-field optical microsocpy, SEM, and AFM. and SEM, microsocpy, optical dark-field microscopy, optical transmission-polarized or reflective- using matrices phage self-assembled the of nanostructures and micro- ( guidelines provide also We . Syringe pump(KDScientific,Syringe no. model 780270) Electroporator (Eppendorf, no. model 2510) PCR machine (Bio-Rad, no. model PTC1148) no.model I2500) Temperature controlled shaker/incubator (New Scientific, Brunswick Incubator 37°C(VWR, no. model 1575) Benchtop (Eppendorf, centrifuge no. model 5424R, cat. no. 22620401) (KurtElectron-beam evaporator J. Lesker, no. model PVD75) no.model JA-20, cat. no. 334831) Fixed-angle rotor centrifuge for 50ml(Beckman Coulter, no.model JLA-8.1000, cat. no. 969328) Fixed- cat. no. 393124) (Beckman Coulter,Centrifuge no. model Avanti J-26XP, medium (Invitrogen,SOC cat. no. 1544034) Deionized water Glycerol (Merck, cat. no. 8.18709.1000) IPTG (isopropyl-b- cat. no. 20-108) (5-bromo-4-chloro-3-indoyl-b-X-gal Agar (Merck, cat. no. 1.01614.1000) cat. no. AU.1000.SL2) Gold-coated Si wafer (100-nm Au, 5-nmTi Technologies, on Si; Platypus angle centrifuge rotor centrifuge angle for 1liter (BeckmanCoulter, d -thiogalactoside; APEX, cat. no. 20-109) Box Box d -galactoside; APEX, 1 ) for the characterization of of characterization the for ) Fig. 6a

– Table

c ).

2

; ref.

1 3 ). © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. completely usingamicrowave andcool to 45°Cbefore use. stored atroom temperature (15–28°C)for upto 6months. Melt thetop agar autoclave. thesolution Divide into 25-mlaliquots. solution This canbe MgCl Top agar inaclean bench.OC contamination makingtheOC. while It isrecommended to make the for subsequent by infection engineered phages; therefore, avoid any phage theculture.result ininsufficientof aeration Overnight culture culture, (OC) a15-mlculture istoo tube narrow. will This 37 °Cfor 12–16hby shakingat225–250r.p.m. 5 LB medium with in5mlof bacteria use a50-mlculture to tube grow colony uninfected asingle XL1-Blue of prepared shouldbe bacteria theday before Step 10andStep 19. 1dbefore, XL1-Blue bacteria Overnight-cultured contamination. chancethe possible of up to 4weeks. 15mm).height: plates The stored canbe for at4°Cinthedark solution. Pour themedium into aPetri dish(diameter: 10mm, autoclave. Cool thesolution to 750 50°C andadd indeionized agar water.11.25 gof Add deionized water upto 750mland IPTG–X-gal pl LBagar at −20°Cfor upto 1year. thesolutionDivide into 0.75-mlportions. solution This stored canbe solution, DMF. in12.5mlof IPTG dissolve X-gal 0.625gof and0.5gof IPTG–X-gal solution the plates upside down to contamination. minimize chance thepossible of plates stored canbe for at4°Cinthedark upto 4weeks. Pour themedium into aPetri dish(diameter: 10mm, 15mm). height: The Cool thesolution to 750 50°Candadd indeionized agar water.of Add deionized water upto 750mlandautoclave. Tetracycline–LB plate agar before use. 1 year. and 2.5 ml deionized of water. This solution can be stored at −20 °C for up to (20 mg/ml), dissolve 100 mg tetracycline in of a mixture 2.5 of ml ethanol of Tetracycline solution REAGENT SETUP • • • • • • • • • • • • • • • • • • • • • • 24-404, 24-412, and 24-430) Pipette tips with filter (20, 200, a cat. no. 25630-250) with baffles Glass 250-mlErlenmeyer(Kimble-Chase, flasks beforeflasks use. Glass 4-liter flasks(Pyrex, cat. no. 4980-4L) before tubes use. the Eppendorf (1.5ml; tubes Eppendorf VW Conical (50ml; tubes Falcon, cat. no. 352070) Round-bottom (14ml; tubes Falcon, cat. no. 352059) Petri dish(100×15mm; Falcon, cat. no. 351029)  (50ml; tubes Centrifuge BeckmanCoulter, cat. no. 334831) for atleast2hbefore use.  (1liter; bottles BeckmanCoulter,Centrifuge cat. no. A98813) cat. no. 28415-505) 25-mm diameter 0.45- (BD,Syringe cat. no. 302830) Micropipette Serological pipettes (VWR, catno. 89130-900) Wafer tweezers scribe glass Diamond-tipped Cylinder compressed of N UV-visible spectrophotometer (BeckmanCoulter, no. model DU800) microscopeOptical (Olympus, no. model IX71) Atomic-force microscope (Asylum Research, no. model MFP-3D) Scanning electron microscope (FEI, no. model Quanta3DFEG) Modular adaptor (CableWholesale, cat. no. 31D1-1640BK) RS232C cable (Parallax, cat. no. 2109245)

CR CR 2 ·6H I I  T T I I

CR CAL CAL Dissolve 25 g of LBMillerDissolve broth, agar, 25gof 7gof and1gof 2 O indeionized water. Add deionized water upto 1liter and I T Autoclave before tubes thecentrifuge use. UVirradiation Sterilize with bottles thecentrifuge I  CAL

CR Thoroughly vortex and homogenize the solution I T 

I To prepare 210mMIPTG–98 (1,000×) mMX-gal CAL To prepare 45 mM (1,000×) tetracycline solution

ate CR µ m polyethersulfone(VWR, (PES) filter I

Store theplates upside down to minimize T

2 Dissolve 18.75 g of MillerDissolve LBbroth and 18.75gof with airblow with gun I Dissolve 18.75 g of MillerDissolve LBbroth and11.25g 18.75gof  CAL R, cat. no. 89000-028)

CR Autoclavebefore theflasks use. nd 1,000 I T I CAL µ µ g/ml tetracycline solution at

Overnight-cultured XL1-Blue l of 1,000×tetracycline solution.l of Autoclave the pipette tips before use. µ © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. l; Genesee Scientific, cat. nos.  

CR

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I CR µ T I T I l of 1,000×IPTG–X-gal l of CAL I I CAL  T I

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and PEG–NaCl solution in isshown before use. 1.6 liters (4-liter flask). in 50ml(250-mlErlenmeyer prepare themedium asneeded; for example, Miller LBbroth 1.25gof water andautoclave. into Divide 5-mlaliquots. For liter-scale amplification, LB medium up to 6months. to 1liter and autoclave. solution This stored canbe atroom temp deionized water. Adjust 1MHCl. thepHto 7.5with Add deionized water up NaCl) solution, Tris-base dissolve 6.06gof NaCl and8.77gof in800mlof TBS buffer room temperature for upto 2months. homogenize theseparatedsolution layers. solution This stored canbe at deionized water upto 1.8liters andautoclave. Shake to mixthesolution and NaCldissolve PEG 8000indeionized water. 262.98gof and 360gof Add PEG–NaCl solution after purification (Steps 36–42). in TBS (Step 32). ( ( saline (TBS) after centrifugation to remove the bacterial debris (Step 31). of precipitated phage pellet (Step 27). ( PEG–NaCl precipitation and removal of supernatant. Inset: magnified image phage supernatant (Step 24). ( centrifugation to precipitate the medium (Step 22). ( phage (supernatant of suspension in LB medium through centrifugation (Step 21). ( bacterial culture (Step 19). ( purification. ( Figure 3 g d a h ) Filtration of the phage suspension to remove aggregates after suspension Bacterial OC debris Centrifuged

| in 800ml M13 phage preparation through liter-scale amplification and phag



To prepare Tris-buffered saline(TBS, 50mM Tris-HCl, 150mM

Dissolve 5 g of MillerDissolve LBbroth deionized in0.2liters 5gof of e CR a Amplified ) Phage amplification in 50 ml of LB medium and additional in 50ml TBS (25ml) Centrifuged phag I phage in T I i ) Highly pure and concentrated phage suspension in TBS CAL e

d To prepare PEG–2.5 20%(wt/vol) MNaCl solution, ) Amplified phage in 800 ml of LB medium after b  Flowchart showing use of LBmedium, Flowchart useof showing TBSbuffer ) through bacterial infection in 1.6 liters of LB

CR natureprotocols h b e suspension Filtered I b phag flask) and 40 g of LB Miller broth in flask)and40g of T ) Separation of I f CAL ) Phage pellet after centrifugation with e E. coli. in 800ml Freshly prepare themedium Phag Figure (LB) E. coli e (Steps 23 and 24). ( g Phage in LB ) Phage suspension in Tris-buffered 4 . E. coli

| VOL.12 NO.9VOL.12 from the phage i f c protocol e in 1.6liters c Amplified ) Decanted ) Amplified | phage Phage pellet 2017

erature for

suspension Purified in TB phage

|

2005 S © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. 4| CAAAAGCGGCCTTTAACTCCC-3 forward primer is 5  site inthe reverse primer tomake the M13plasmid linear byPCR(reverse primer: 5 PstI restriction site(CTGCAG, inbold)the forward primer, followed bythe desired insert sequence. Add the PstIrestriction 3| manufacturer’s instructions. 2| 5 45 basesinlength, withamelting temperature (  1| or wiping down with70%(vol/vol)ethanol and atleast2hof UVirradiation before use  Genetic engineeringof phage PROCE (which is dependent on the the of pulling routine. pulling speed) (i.e., up vertically or down), speed (i.e., 0.1 program (available from the authors upon request), which sets the direction pump.syringe pump The is syringe controlled a with custom computer direction, respect with to the horizontal plane, on the the of moving part adaptor ( pump connected syringe to a computer an with RS232C cable and a modular Computer-controlled device pulling EQUIPMENT SETUP to supernatant is 1:6. purification. For all PEG–NaCl precipitation steps, theFigure target 4 ratio of PEG–NaCl 2006 phage inLB Total Nuclease-free water DMSO 5× Phusion Buffer dNTPs Primers Phusion high-fidelity DNA polymerase M13KE vector C protocol ′ -CAAGCTTGCATGCCAGCAGGTCCTCGAATTCACTG-3

omponent 3 ×1.6 Step 22

CR CR CR

Prepare the PCRmixasfollows using the primers designed inStep3: Design forward and reverse synthetic oligonucleotide primers for the genetic modification of the pVIIIprotein. Add the Change T6246toA6246inthe M13KEvectorusing the QuikChange IIsite-directed mutagenesis kitbyfollowing the Design mutagenic oligonucleotide primers tomodify the PstIrestriction site(CTGCAG) in | I I I VOL.12 NO.9VOL.12 T T T D

| l Fig. I I I The workflow for phage PEG precipitation steps in phage URE CAL CAL CAL Steps 23and24 5 phage inLB ). The tweezers are lengthwise installed along the vertical Sterilization consists of either autoclaving and storing inadry place(preferably anovenat<70°C),

6 ×0.8 STEP STEP | Add 133ml PEG–NaCl l 2017 Inthe caseof quaternary glutamate (EEEE,4E)insertion into the pVIIIprotein, the sequence of the Foreffectivemutagenesis reaction, mutagenic oligonucleotide primers should bebetween25 and Step 25 ′ -ATATAT phage inTBS 6 ×25ml | Step 28

natureprotocols CT ′ (desired sequence is in italics, R = A/G). PEG–NaCl Add 4.2ml G Step 33

CA The pulling device The is pulling device a motorized phage inTBS ● G 10 Step 35 µ

© 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. AAGAAGAGGAA T m/min–30 m/min–30 mm/min), and time × IMI

1 ml PEG–NaCl Add 167 N Step 37 G phage inTBS 3d 2% 2% (vol/vol) 4 ×1ml Step 39 0.5 0.5 Up Up to 50 µ l A 200 200 100 100 ng ′ mount 50 50 10 and 5 µ 1 1 U CCCGCAAAAGCGGCCTTTAACTCCC-3 M M each T µ µ m µ l l M ) of Final phage µ ′ Step 42 l -CAGTGAATTCGAGGACCTGCTGGCATGCAAGCTTG-3 stock ≥

78 °C.The recommended sequences of primers are

substrate from phage suspensions. substrate the holders. of process The exhibits pulling photograph enlarged The of device consists a computer, and syringe pump, substrate speeds. controlled at phage suspensions precisely concentrated from pure highly and substrates pull device pulling to vertically by onto using a a computer-controlled substrate Phage is self-assembled 5 Figure

| Photographs of computer-controlled pulling device.pulling ofPhotographs computer-controlled ′ or 5 ′ -CACCCT ′ - ATATAT 6 7 . CT CT G LacZ CA G CA G α ′ CGAAAGACAGC-3 (M13KE). . G ARGARGARGAR Meniscu

s

CCCG Pulling

′

). suspensions Phage

Substrat

- e © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. fuge tube(1.5 ml)and centrifuge the tubeat18.4k 14| 10  on anorbitalshaker operating at225r.p.m. tube. Culture the tubeovernight, orfor 8–16hat37°C, 1.5 mlof LBmedium ina14-mlround-bottom culture plate inStep10)and 15 well-separated blueplaquefrom anIPTG–X-gal screening 13| T Verification of a clonal population of mutant phage using a−96gIIIsequencing primer (5 12| and identification of phage. phage. Onlywell-separated blueplaques(i.e., each plaquecontains asingle typeof phage) are picked for further culture   OC XL-1Bluebacteria at37°Cfor ~4–6h. 11| and pouritwith3mlof topagarose onto IPTG–X-gal LBagarplatesand incubate them at37°C. 10| pected efficiency depends on the inserted peptide sequence; efficiency is tion cuvette, and immediately add 950  37 °Cbefore use)at37°Cfor 1hbyshaking at225–250r.p.m. the manufacturer’s instructions). Incubate the transformed XL1-Blue cellsin950 electroporation-competent cellsand transform atavoltage setting of 1,700Vbythe electroporation method (according to 9| vector plasmid (all the purified vector from Step 7) and T4 DNA ligase (200 units) and incubate the mixture at 16 °C for 18 h. 8| PstI-digested M13KEvectorusing aQIAquick PCRPurification Kit(according tothe manufacturer’s instructions). fied product, and incubate the mixture for 4hat37°C.Inactivate PstIactivitybyincubation at80°C for 20min.Purifythe 7| (size ~7.3kb)using aQIAquick GelExtraction Kit(according tothe manufacturer’s instructions). 6|  5| 4 °C( 33 32 2–31 1 C IMI

ycle no.

11 PAUSE CR CR CR CR Extract the engineered phage vectorfrom Pick engineered phage plaques from the platesand culture eachplaquein1mlof LBmedium containing 10 Mix1–20 Transfer the contents of the culture tubetoacentri Add 15 virusperml)of phage stock ispreferred. N For optimal efficiency of the electrotransformation, add 1 Perform arestriction digestion of the PCRproduct bymixing the PstIrestriction enzyme (40U)with100 Separate the PCRproduct using gel electrophoresis witha0.6%(wt/vol)agarose gel. Extract and purifythe PCRproduct Use the following PCRprogram: Ligate the PstI-digested M13KE vector plasmid for recircularization with T4 DNA ligase. Mix 10 I I I I T T G T T T able I I I I 2 d CAL CAL CAL CAL

PO

µ 3

I STEP STEP STEP STEP NT ) for 10minto pelletthe bacteria. l of phage stockfrom Step 11(orasingle, µ 98 98 °C, 1 min l of the transformed bacteria with200 98 98 °C, 15 s The PCR product can be stored at −20 °C until you are ready for Step 6. Denature To increase the transformation efficiency, transfer the competent cell–DNA mixture to a prechilled electropora The blueplaqueformation onIPTG–X-gal LBagar platesisthe result of growing agenetically modified M13KEcontains the Aconcentration of >0.01mg/ml (3.2× µ l of OCXL-1Bluebacteria to 60 60 °C, 30 s A © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. nneal ′ µ -CCCTCATAGTTAGCGTAACG-3 LacZ l of SOC medium (prewarmed to 37 °C) after electroporation to resuspend the cells. Ex gene, sothe infectedplaques appearblueonthe IPTG–X-gal plate. 72 72 °C, 5 min 72 °C, 5 min E. coli E xtend using aQIAprep SpinMiniprep Kit.Confirmthe inserted sequences g at µ ● l of overnight-cultured XL-1Bluebacteria (Reagent Setup), -

pH 7.5;150mMNaCl. Note thatthese phage structures canbeobtained using the following bufferconditions: 50mM Tris, phage hierarchical structures. T able able Smectic helicoidal nanofilament Cholesteric helical ribbon Nematic orthogonal twist Nematic stripe pattern P µ hage structure 4 4 °C, hold 4 °C, hold l of the ligation product to50 ′ Final ) byDNAsequencing. 2 2 |

The fabrication conditions of varying representative ≥ 1 × 10 8 plaque-forming units per µ l of SOCmedium (prewarmed to natureprotocols P hage conc. µ (mg/ml) 0.25–0.5 0.2–1.5 0.1–0.2 l of XL1-Blue 4–6 µ l of PstI-digested M13KE

| VOL.12 NO.9VOL.12

µ g for M13KE DNA. P ulling speed ( protocol

µ m 10–120 30–120 10–120 10–20 l of the puri m/min) µ |

l of 2017

|

2007

- - - © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. desired mutation and also to evaluate wild-type or mutation contamination by checking for the existence of minor DNA peaks. sequencing (Steps14–17) ( 23| P IPTG–X-gal LBagarplates(Step10)). in LBmedium canbedetermined bytitration (i.e., counting blueplaqueson serial-diluted phage-incubated in 50-mlscalecultures original sequence, isoften observedduring thisstep. Typically, phage titersreach upto10 to the LBmedium. It isimportant tonote thatwild-typecontamination, aswellmutations of the contact withthe sides of the flasks. Liquid containing amplifiedphage and bacteria should bedirectly transferred  for ~12–16hat37°Cand 225r.p.m. ( (from Step21)and 14mlof fresh uninfectedOCXL1 Bluebacteria (from Step19).Incubate the flasksbyshaking them 22| at 4°C( T 21| 20| for 5–9hat37°Cbyshaking at225r.p.m. ( In the second flask,grow 50ml of uninfectedbacterial culture byadding 500 19| 18| 4-liter flasks. contamination isnecessary aftereachtransferring stepof the medium. Thisprotocol describes the useof three separate increases of medium from small tolarge scaleare recommended. Performance of phage genome identification toevaluate  L the contamination bychecking the existence of minor DNApeaks. the cloning sites(gIIIand gVIII)toensure thatyourphage particle contains the desired mutation and alsotoevaluate  sequencing. The remaining double-stranded phage DNAcanbestored for genetic engineering for upto1yearat−20°C. 17| 16| glycerol can be added and the phage stock can be stored at −20 °C for up to 5 or more years.  on serial-diluted-phage-incubated IPTG–X-gal LB agar plates (Step 10)). Keep the The typical phage concentration of the supernatant is ~0.01–0.2 mg/ml, as determined by titration (i.e., counting15| blue plaques 2008 able able 15.9 15.9 k 12.1 k 18.4 k g protocol iter-scale amplification iter-scale amplification hage hage purification

PAUSE CR CR CR CR Transfer the upper 90% of the phage-containing supernatant to a fresh centrifuge tube and discard the remaining 10%. Dispense the culture evenlyinto sterilized1-litercentrifuge bottles, keeping 1mlof cultured solution for DNA To three separate 4-literflaskscontaining 1.6liters of LB medium each,add 14ml of amplified phage supernatant Divide the remaining phage-infected culture into two50-mlcentrifuge tubesand centrifuge them for 20minat12.1k Remove 1mlof phage-infected culture for DNAsequencing (Steps14–17). Transfer 500 Prepare two250-mlbaffledflaskswith50ml ofLB mediumeach. Evaluate the phage genome sequences using −96gIIIsequencing primer (5 Extract the double-stranded DNAfrom the bacterial pelletusing aQIAprep SpinMiniprep Kit.

| I I I I VOL.12 NO.9VOL.12 T 3 3 T T T I I I I CAL | CAL CAL CAL T

Centrifugal Centrifugal speeds. able PO

Depending onthe experimental setup,the scaleof phage amplification canbevaried. However, incremental

I STEP STEP STEP NT 14, 14, 36, 38, 40–42, and 44 3 The phage stock can be stored at 4 °C for several months. For longer storage, the same volume of sterile ; | It is important to sequence both cloning sites (gIII and gVIII) to ensure that your phage particle contains the µ 2017 Fig. 3 When solutions are transferred, the serological pipetteand solutions must not come into The −96gIIIprimer coversDNAsequencing of the gIIIand gVIIIsites. It isimportant tosequence both l of phage stocksolution from Step15and 500 24, 24, 26, and 27 21, 31, and 34 ●

1 | T S

6 b IMI natureprotocols teps , whereas lowertitersare observedin1-literorlarger-scale cultures. The phage concentration ). Decant the supernatant into new tubeswithout disturbing the bacterial pellet. ● N Fig. 3

G T 2 d IMI d © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. N ). G 2 d Fig. 3 r.p.m. c Fig. 3 10 10 k 14 k ). 8 8 k a ). Beckman Coulter, Avanti J-26 XP Eppendorf, 5424R C entrifuge (company, modelno.) µ l of OCXL-1Bluetoone of the 250-mlbaffledflasks. µ ′ l of OCXL-1Blue. Culture the flasks -CCCTCATAGTTAGCGTAACG-3 E. coli pellet for DNA sequence analysis. 13 Beckman Coulter, JLA-8.1 Beckman Coulter, JA-20 Eppendorf, FA-45-24-11 R viral particles perml otor (company, modelno.)

′ ) byDNA

g

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. 38|  37| tubes. The pelletnow contains the remaining bacteria orPEGand phage aggregates. Discard the pellet. 36|  and vortex the tubestohomogenize. Divide the homogenized suspension into ten1.5-mlcentrifuge tubes. 35| ?  lect allphage atthe bottomof the tubes. Discard the remaining supernatant. 34| 33| ? (25-mm diameter inoursetting) ( 32| or PEGand phage aggregates. Discard the pellet. 31| 30| repeatedly tomake ahomogeneous phage suspension.  previous 10mlof phage solution inTBSand vortex tohomogenize the solution. up and down tocapture asmany phage particles aspossible. Transfer the solution to50-mlcentrifuge tubeswiththe 29| PBS canalsobeused(e.g., for chemical modification).  ensure ahomogeneous suspension of phage particles. Transfer the solution tonew 50-mlcentrifuge tubesand vortex. phage pellet.Pipettethe solution upand down tobreak upthe remaining clumps. Agitate the bottleagainfor 10minto 28| ?  the precipitated phage atthe bottomof the bottles. Discard the remaining supernatant ( 27| 26| we havefound thatcentrifugation isoften the critical stepduring PEGprecipitation. for phage PEGprecipitation stepsisshown in  mix and incubate them at4°Cfor 12–16h. 25| centrifuge bottles. Eachbottleshould haveafinal volume of 800ml of clearphage supernatant ( 24|

TROUBLES TROUBLES TROUBLES

CR CR CR CR CR CR CR Centrifuge the tubesfor 10minat18.4 k Add 167 Centrifuge the tubesfor 20minat18.4k Resuspend the pelletsfrom six50-mlcentrifuge tubesin10mlof TBS,pipettethe suspension upand down, Centrifuge the tubesfor 20minat12.1k Add 4.2mlof PEG–NaCl toeach25mlof phage-containing supernatant, and incubate the tubesat4°Cfor ~12–16h. Filterthe supernatant from Step31using a0.45- Centrifuge the tubesfor 10minat12.1k Incubate the phage solution in50-mlcentrifuge tubes at4°Cfor 5h. Add anadditional 15ml of TBStothe phage pelletinthe original 1-litercentrifuge bottle. Pipettethe solution Add 10mlof TBSsolution toeachbottle. Closethe bottleand agitateitfor 10mintobreak upand resuspend the Remove allof the supernatant and centrifuge the bottlesagainfor 15minat15.9k Centrifuge the bottlesfor 20minat15.9k Add 133mlof PEG–NaCl solution toeach1-litercentrifuge bottletoafinal volume of 0.933liters. Shake the bottlesto Centrifuge for 20minat15.9k I I I I I I I T T T T T T T I I I I I I I CAL CAL CAL CAL CAL CAL CAL

H H H STEP STEP STEP STEP STEP STEP STEP µ OOT OOT OOT l of PEG–NaCl toeachtube and incubate the tubesat 4°Cfor ~1.5–2h. To break up clumps, it is important to vortex the tubes and pipette the suspension up and down vigorously. The pelletof the phage inthisstepappearsopaque white. To break upclumps, itiscritical tovortex the tubesvigorously and pipettethe solution upand down TBShasbeenwidely usedasabufferfor M13bacteriophage toobtainthe beststability. Alternatively, The pelletof the phage atthisstepappearsopaquewhite. ForallPEG–NaCl precipitation steps, the target ratio of PEG–NaCl tosupernatant is1:6.The workflow Incubate the tubesfor <2h.Longer incubation will affectthe quality of the phage film. I I I N N N G G G © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. Fig. 3 g at4°C( h ). Figure g g g g T at4°C( at4°C( at4°C( at4°C( able g at4°C( 3 4 µ ) topelletthe bacteria. Decant the supernatant into 1-liter . There are anumber of factors thatcontribute tothe overall yield; m polyethersulfone (PES)-membrane syringe filter T T T T able able able able T able 3 3 3 3 ) topelletthe phage and discard the supernatant. ). Transfer the supernatant totenclean1.5-mlcentrifuge ). Discard the supernatant. Centrifuge one more time tocol ; 3 Fig. 3 ). g ). The pelletnow contains the remaining bacteria natureprotocols Fig. 3 g at4°C( f ). Fig. 3d T able

| VOL.12 NO.9VOL.12 ,

e 3 ). ) toconcentrate protocol

|

2017

|

2009

- © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. for representative phage nanostructures are described in cholesteric helical ribbon,and smectic helicoidal nanofilament structures, canbe fabricated. The self-assembly conditions chemistry, and substrate surface, avariety of self-assembledstructures, such asnematic stripe, nematic orthogonal twist,  Depending onthe parameters, the resulting phage structures should bedifferent ( 52| 51| aggregated PEG,and phage. 50| we prepare ~1mlof the phage suspension (withTBSsolution for color-phage sensor). 49|  48| (Equipment Setup; 47| 46| using adiamond-tipped glassscribe. 45| S  Prepare the desired concentration of the phage suspension in a desired buffer system for self-assembly experiments ( 44| ? mg/ml phage concentration. To prevent analytical error, a plaque-forming assay should be used periodically to ensure accuracy. 100: dilution factor, UV-visible spectroscopy using equation (1). 43| 1.5-ml centrifuge tubes( 42| supernatant toclean1.5-mlcentrifuge tubes. 41| The pelletnow contains the remaining bacteria orPEGand phage aggregates. Discard the pellet. 40| ensure thatthe genome iscorrect, and itdoes not contain any mutations. buffer canbevaried according tothe purposeof the subsequent experiments. It isimperative tosequence the phage to  phage solution willbe10mg/ml inTBS. to homogenize. Divide the homogenized suspension into four 1.5-mlcentrifuge tubes. The target concentration of the final 39| 2010 protocol elf-assembly elf-assembly

TROUBLES

PAUSE CR CR CR CR Once you have a final concentration of phage in solution, verify the phage genome using DNA sequencing (Steps 14–17). Runthe designed pulling program. The filmshould bepulledvertically ata speedbetween~10and 300 Lowerand dipthe waferinto the phage solution. Before the self-assembly process, centrifuge the phage suspensions for 10mintoremove any remaining bacteria, Prepare highly pure phage suspensions atthe desired concentrations ( Mount the gold-coated Siwaferonthe setup. To accomplishthis, weuseapreprogrammed syringe pump(KDScientific) controlled through anRS232Ccable Cleanthe waferbyblowing itwithnitrogen gas. Prepare the desired substrates. Forexample, cutagold-coated Siwafertothe desired size(typically 2×0.5cm) Dilutethe phage-containing solution 100times withTBSbuffer. Measure the concentration of phage particles with Centrifuge the supernatant for 20minat18.4k Incubate the supernatant for 1hat4°C,and centrifuge itfor 20minat18.4k Centrifuge the tubefor 20minat18.4k Resuspend the combined pelletsof 10tubesin4mlof TBS,pipettethe suspension upand down, and vortex the tube

| I I I I VOL.12 NO.9VOL.12 T T T T I I I I CAL CAL CAL CAL

PO I

H NT STEP STEP STEP STEP OOT Purified phages can be stored for 1 year at 4 °C. Before use, centrifuge the stock for 20 min at 18.4 k ● |

OD represents the optical densities at 269 and 320 nm, respectively. Usually, 4 ml of dispersion has a 6 –10 2017 Bytuning parameters such asphage concentration, pulling speed, ionic concentration, phage surface Undesired movement of the substrate willproduce substantial defects during the self-assemblyprocess. The required resuspension volume canbedetermined bythe pelletsize. Inaddition, the final dispersion I T N IMI Fig. G A 269 N |

5 natureprotocols G : absorbance at269nmfor M13phage, Fig. 3 ). Setthe program according tothe vendor’s instructions. 1–24 h, depending on the pulling speed i ). © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved. mg of g at4°C( phage/ml g at4°C( T × = T able able 100 3 2 T ). Transfer the supernatant toaclean1.5-mlcentrifuge tube. able (A . A 320 269 : absorbance at320nmfor background. 3 − ). Transfer the supernatant toclean ) A 320 T able /3.8 2 T ) in1.5-mlcentrifuge tubes. Typically, 4 able

g at4°C( 2 ). ) ( 1 T able 3 ). Transfer the

g at 4 °C µ m/min. (

T able T able

2 3

(1) ). ).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. phage hierarchical structures are described in pulling speed, and ionic concentration) for representative The detailed fabrication conditions (concentration of phage, structures canbefabricated, asshown in orthogonal twist,and smectic helicoidal nanofilament such asnematic stripe, cholesteric helical ribbon,nematic and substrate surface, avariety of self-assembledstructures pulling speed, ionic concentration, phage surface chemistry, By tuning various parameters, such asphage concentration, assembly process occurring atthe meniscus ( from the phage suspensions, leading toaself-templating the self-assemblyprocess, the substrate isslowlypulled reproducible preparation of high-quality phage films. During amplifications. Ahighly purifiedphage solution is keyto 120 mg of phage are typically obtained from 4.8-literscaled numbers of the desired phage; final yields between 10and is possible. The M13phage replicates and produces large sequence-verified clone, liter-scaleamplification ofphage genetically engineered phage stocksolution. Using a protocol, one should successfully obtainahighly pure Upon completion of the experiment described inthis ANT B pulling speeds Steps 45–53,self-assembly:1–24h,depending onthe Steps 23–44,phage purification: 2d Steps 18–22,liter-scaleamplification: 2d phage: 2d Steps 13–17,verification of aclonal population of mutant Steps 1–12,genetic engineering of phage: 3d ● Troubleshooting advice canbefound in ? shifted toward the bluevisiblespectrum( increase of pulling speed, the colorsoneachlayer are phage/ml and bysetting the pulling speedfrom 10to120 53| T (ref. able able 43 32 27, 34 S

ox 1 TROUBLES

tep T IMI (Optional) Fabricate bundled phage nanofilaments that exhibit different colorsbyusing asolution of 4–6 mg of I 1 C , filmcharacterization: 2–5h 3 I 4 4 PATE N ). These nanostructured phage films canbeused for | G

Troubleshooting table. D phage is off The concentration of the the filter well The sample does not go through white phage pellets pellet has formed on the opaque A translucent white precipitated P H roblem OOT RESULTS I N G © 2017 Macmillan Publishers ofSpringer Limited, part Nature. All rightsreserved.

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Fig. 2 Fig. T 4 able initial stock solution, and so on type of phage, the concentration of phage can vary depending on the The final concentration of the too high The phage concentration may be precipitated Bacterial cell debris may have P . a ossible reason 6 ). 5 .

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m/min with10- with permission from ref. and right-handed helicoidal nanofilaments (scale bar, 5 50 (cross-polar) of smectic helicoidal nanofilament (SHN) structure (scale bar, respectively (scale bars, 100 structure on the left and right sides, which show right- and left-handedness,the rolling of phage supramolecular structures. (Top) SEM images of the CHRhelical ribbon (CHR) structure (scale bar, 300 progressing from a groove to ridge area. ( pulling direction. Left-handed rotation of phage-fiber bundles occurs when (scale bar, 5 Inset shows an AFM image of the phage-fiber bundled structures on a ridge ( 5 an AFM image of the phage-fiber bundled structures on a ridge (scale bar, ( Figure 6 b a µ a c ) Photograph of nematic striped pattern (scale bar, 100 ) Photograph of a nematic orthogonal-twist structure (scale bar, 100 µ m). Phage fibers on a ridge were aligned to the pulling direction. ridge Groove m). Inset: AFM image exhibited SHN structure composed of left-handed Right-handed

| Representative examples of self-assembled phage nanostructures. µ

m). Phage fibers on a ridge were perpendicularly aligned to the µ m/min increments. Withthe incremental adjusting the volumes accordingly trate with a PEG precipitation (Steps 37–42), If the final concentration is too low, concen Use a fresh filter pellets only 10 ml of TBS. Collect the opaque white phage remove the translucent part by washing it with To improve the purity of the phage solution, S olution Left-handed 1 natureprotocols 3 , Nature Publishing Group. µ m). ( Ridge d ) Polarized optical microscopy image b d c , bottom) Photograph of cholesteric ridge Groove µ m). Curved meniscus induces

| VOL.12 NO.9VOL.12 µ m). Image adapted µ protocol m). Inset shows | 2017

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- m). 2011

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. regard to jurisdictional claims in published maps and institutional affiliations. com/reprints/index.htm at online available is information permissions and Reprints interests. CO procedure for the film-characteristics experiment involved J.H.L. and S.-W.L. The experiments on phage involved self-assembly J.H.L., C.M.W., and S.-W.L. The S.-W.L. were made with regard to liter-scale amplification and phage purification. S.-W.L. Similar contributions by J.H.L., C.M.W., H.-E.J., E.B., A.R.P., E.J.P., and population of mutant phage were designed or performed by J.H.L., H.-E.J., and experiments on genetic engineering of the phage and verification of a clonal and all other authors provided substantial editorial revisions. Representative AUT (2016R1C1B1008824). H.-E.J. is supported by a NRF grant funded by the Korea government (MSIP) of Korea (NRF; and 2016K2A9A1A01951919 NRF-2014S1A2A2027641). cooperation program managed by the National Research Foundation Program. This work was supported under the framework of international supported in part by the Samsung Electronics Global Research Outreach support from the Tsinghua-Berkeley Shenzhen Institute. This work was also Chief of Engineers to publish this information. We acknowledge funding Quality Research Program Permission(611102T2500). was granted by the A self-templating assemblyapproaches. next-generation functional materials withbiomimetic far-from-equilibrium structures. We canalsodesign decipher the natural self-assemblyprocesses tobuild parameters of the phage self-assemblyprocess, wecan investigation of the governing thermodynamic and kinetic tissue regeneration piezoelectric energy harvesting, colorimetric sensors, and 2012 14. protocol ckno

M

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