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Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk Christopher H. Bowen, Bin Dai, Cameron J. Sargent, Wenqin Bai, Pranay Ladiwala, Huibao Feng, Wenwen Huang, David L Kaplan, Jonathan M. Galazka, and Fuzhong Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00980 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 7, 2018
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Biomacromolecules
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TITLE: Recombinant Spidroins Fully Replicate Primary Mechanical
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Properties of Natural Spider Silk
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AUTHORS: Christopher H. Bowen1,†, Bin Dai1,†, Cameron J. Sargent2, Wenqin Bai1, Pranay
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Ladiwala1, Huibao Feng1, Wenwen Huang4, David L. Kaplan4, Jonathan M. Galazka5, Fuzhong
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Zhang1-3,*
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AFFILIATIONS:
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1
Department of Energy, Environmental and Chemical Engineering,
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2
Division of Biological & Biomedical Sciences,
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3
Institute of Materials Science & Engineering,
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Washington University in St. Louis, Saint Louis, MO 63130, USA
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4
Department of Biomedical Engineering, Tufts University, Medford, MA 02155, USA
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5
Space Biosciences Division, Ames Research Center, National Aeronautics and Space
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Administration, Moffett Field, CA 94035, USA
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†
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*
These authors contributed equally. Correspondence to:
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ABSTRACT:
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Despite significant efforts to engineer their heterologous production, recombinant spider
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silk proteins (spidroins) have yet to replicate the unparalleled combination of high strength and
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toughness exhibited by natural spider silks, preventing their use in numerous mechanically-
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demanding applications. To overcome this long-standing challenge, we have developed a
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synthetic biology approach combining standardized DNA part assembly and split intein-
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mediated ligation to produce recombinant spidroins of previously unobtainable size (556 kDa),
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containing 192 repeat motifs of the Nephila clavipes dragline spidroin. Fibers spun from our
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synthetic spidroins are the first to fully replicate the mechanical performance of their natural
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counterparts by all common metrics, i.e. tensile strength (1.03 ± 0.11 GPa), modulus (13.7 ± 3.0
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GPa), extensibility (18 ± 6%), and toughness (114 ± 51 MJ/m3). The developed process reveals a
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path to more dependable production of high-performance silks for mechanically-demanding
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applications while also providing a platform to facilitate production of other high-performance
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natural materials.
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INTRODUCTION:
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Dragline spider silk is among the strongest and toughest biological materials, capable of
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outperforming most synthetic polymers and even some metal alloys.1-3 These remarkable
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properties have resulted in a growing list of potential high-performance material applications for
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spider silks ranging from impact resistant textiles to monofilament surgical sutures;
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unfortunately, the impracticalities of spider farming have long restricted the practical
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development of such applications.2,4 The resulting demand for a dependable source of high-
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performance silk fibers has driven both academic and industrial research toward production of
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recombinant spidroins in engineered hosts including bacteria, yeasts, plants, and animals.5-9
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While providing significant technical and scientific advances, these efforts have so far been
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unable to yield recombinant silk fibers that fully replicate natural spider silk's unparalleled
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combination of mechanical properties (i.e. high strength, modulus, extensibility, and toughness),
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due in part to an inability to stably produce highly repetitive, high molecular weight (MW)
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proteins in heterologous hosts.2,4,9
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Natural dragline spidroins are typically very large (>300 kDa), highly repetitive proteins
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that contain hundreds of tandem repeats of glycine- and alanine-rich sequences.10,11 As with most
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polymers, the size of these spidroins is expected to positively correlate with tensile strength (up
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to a limiting MW) due to an increased density of interchain interactions and entanglements and
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fewer chain-end defects.9,12 Indeed, previous work has demonstrated a clear correlation between
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MW and strength for recombinant Nephila clavipes dragline fibers, with the largest spidroin (96-
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mer, 285 kDa) yielding the strongest recombinant fiber reported to date (σ ≈ 550 MPa).9
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However, the upper limit for the strength-to-MW relationship in spidroins is currently unclear
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and despite the apparent need for even larger spidroins to yield fibers with mechanical properties
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on par with natural spider silk (σ = 0.8-1.2 GPa for N. clavipes dragline, see Table S1),
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heterologous production of spidroins larger than 285 kDa in quantities sufficient for fiber testing
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has proven to be extremely difficult.9 Challenges of heterologous production arise from a series
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of issues including genetic instability of long and highly repetitive DNA sequences, translation
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inhibition by complex mRNA secondary structures, a high demand for glycine and alanine
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tRNAs, and overall metabolic burden caused by spidroin overexpression.4,9 To overcome these
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long-standing challenges, we envisioned a synthetic biology platform employing standardized
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DNA part assembly to facilitate covalent, post-translational ligation of the largest spidroins that
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can be stably expressed in engineered Escherichia coli (i.e. 96-mer), permitting production of
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spidroins of previously unobtainable MW that can be used to generate fibers with exceptional
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mechanical properties (Figure 1).
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Of the various biochemical approaches available for post-translational protein ligation,
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split inteins (SIs) are perhaps the best suited for generating high MW proteins for material
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applications. SIs are peptide auto-processing domains that, when fused to separately expressed
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proteins, catalyze spontaneous splicing reactions, covalently linking their fusion partners via a
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peptide bond and leaving only a few residues (≤ 6) at the ligation site.13 When SIs are used to
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ligate 96-mer spidroins, these residues are unlikely to affect the properties of the much larger
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ligated product (6720 amino acids total). Other common approaches for post translational
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ligation involve either side chain bonding (e.g. SpyTag-SpyCatcher), which produces branched
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structures that could negatively affect material properties, or catalysis by separately expressed
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proteins (e.g. Sortase A), which increases process complexity.14 For these reasons, we chose to
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employ SI-based ligations for our envisioned platform, such that ligation of an N-intein (IntN)-
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fused 96-mer spidroin (96N) with a C-intein (IntC)-fused 96-mer (C96) would yield a 556 kDa,
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192-mer spidroin (Figure 1e).
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Figure 1. Process schematic for split intein-mediated ligation of spider silk proteins with
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unprecedented molecular weight and mechanical properties. (a) The highly repetitive core of
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natural N. clavipes dragline silk protein MaSp1 (shown as a simplified consensus peptide
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sequence) was reduced to a single repeat unit (1-mer). Note, while "-mer" often refers to a small
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molecule or individual amino acid, the abbreviation here refers to the 35 amino acid peptide that
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constitutes the repeating unit of the larger spidroin. (b) The 1-mer DNA sequence was combined
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in silico with 5' UTR, RBS, split intein (SI), and terminator sequences, which were then
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computationally optimized for microbial production. (c) The optimized DNA sequences were
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assembled within the framework of a standardized DNA part assembly system termed SI-Bricks
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to yield complementary IntC- or IntN-fused 96-mer constructs. (d) Constructs were transformed
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to metabolically engineered E. coli for bioproduction. (e) Cell cultures were mixed and lysed to
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initiate SI-mediated covalent ligation of 96-mer spidroins to yield a 192-mer, 556 kDa product.
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(f) The ligated product was purified and spun into fibers for mechanical testing. Scale bar
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indicates 5 µm.
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MATERIALS AND METHODS:
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Strains and growth conditions. E. coli NEB 10-beta (NEB10β) was used for all plasmid
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cloning and protein production. For all cloning, E. coli strains were cultured in Terrific Broth
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(TB) containing 24 g/L yeast extract, 20 g/L tryptone, 0.4% v/v glycerol, 17 mM KH2PO4, and
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72 mM K2HPO4 at 37 °C with appropriate antibiotics (50 µg/mL kanamycin and 30 µg/mL
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chloramphenicol). M9 glucose medium with tryptone supplement (2% w/v glucose, 1x M9 Salts,
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75 mM MOPS pH 7.4, 12 g/L tryptone, 5 mM sodium citrate, 2 mM MgSO4·7H2O, 100 µM
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FeSO4·7H2O, 100 µM CaCl2·2H2O, 3 µM thiamine, 1x micronutrients [40 µM ZnSO4·7H2O, 20
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µM CuSO4·5H2O, 10 µM MnCl2·4H2O, 4 µM H3BO3, 0.4 µM (NH4)6Mo7O24·4H2O, and 0.3 µM
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CoCl2·6H2O]) was used for protein production in bioreactors.
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Chemicals and reagents. Unless otherwise noted, all chemicals and reagents were
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obtained from MilliporeSigma. Plasmid purification and gel extraction kits were purchased from
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iNtRON Biotechnology. FastDigest restriction enzymes and T4 DNA ligase were purchased
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from Thermo Fisher Scientific and used for all digestions and ligations following the
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manufacturer's suggested protocols.
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Genetic assembly of 64- and 96-mer spidroins. The multimeric spidroin DNA
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sequences were constructed as SI-Bricks genetic parts based on a method modified from
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previous work (Supplementary Note 1, Figure S1, Figure S2).5,9 Specifically, an optimized
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coding sequence of 1-mer N. clavipes MaSp1 was chemically synthesized by Integrated DNA
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Technologies. To fit the SI-Bricks standard, the DNA sequence was flanked on the 5' end by
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restriction sites 5'-KpnI/NheI-3' and on the 3' end by restriction sites 5'-SpeI/Kpn2I-3'. The
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sequence was inserted between restriction sites KpnI/Kpn2I of a medium copy (pBBR1
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replication origin), chloramphenicol resistance (CmR) SI-Bricks expression vector, resulting in
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plasmid p1 (Figure S2b). To begin the recursive directional genetic assembly, plasmid p1 was
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linearized by digestion with NheI and ligated with another 1-mer sequence digested by
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NheI/SpeI. The ligation was transformed to NEB10β for amplification, yielding plasmid p2
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containing 2-mer spidroin (Figure S2c). The same procedure was repeated for p2, with insertion
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of a linearized 2-mer sequence, to yield plasmid p4, and the process was repeated until p64 (32-
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mer + 32-mer) and p96 (64-mer + 32-mer) were obtained (Figure S2d). Because the annealing of
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NheI/SpeI overhangs produces a non-cutting sequence, digestion with NheI was used to confirm
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proper insert direction after each ligation iteration.
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Construction and sequence optimization of silk-SI-fusion proteins. N- and C-intein
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amino acid sequences (CfaN and CfaC, respectively) were obtained from a recent publication15
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and prepared as SI-Bricks standard parts as described below. To ensure substantial production of
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the final SI-fused silk proteins, the SI coding sequences were optimized for E. coli expression
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within the genetic context of all flanking sequences using a combination of computational
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approaches.16,17 Specifically, an initial DNA sequence was computationally designed that
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contains (from 5’ to 3’) a 5' UTR/RBS containing the EcoRI/BglII sites, a short coding sequence
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(5'-ATGGCTAAGACTAAA-3') intended to increase translation initiation rate as described
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previously,18 the CfaC coding sequence, the KpnI/NheI sites, and the multimeric silk sequence.
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Within this genetic context, only the CfaC sequence was set variable and optimized using a
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modified E. coli codon usage table with extra weight given to 5' mRNA structure minimization
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within the sequence optimization algorithm.19 The resulting optimized CfaC sequence (including
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5' UTR/RBS and necessary SI-Bricks restriction sites; Table S2) was synthesized as a gblock
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fragment by Integrated DNA Technologies and was inserted 5' of the 64-mer or 96-mer
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sequences by digestion/ligation with EcoRI/KpnI to yield plasmids pC64 and pC96, which encode
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fusion proteins C64 and C96, respectively (Table S3, Figure S2e). Similarly, to codon-optimize
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CfaN, an initial DNA sequence was computationally designed containing (from 5’ to 3’) the
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multimeric silk sequence, the SpeI/Kpn2I restriction sites, a CfaN coding sequence, and the
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BamHI/XhoI sites. Within this context, only the CfaN coding sequence was subjected to the
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optimization process. The resulting CfaN sequence (Table S2), including SI-Bricks restriction
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sites, was synthesized and inserted 3' of the 64-mer and 96-mer sequences by digestion/ligation
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with Kpn2I/XhoI to yield plasmids p64N and p96N, which encode fusion proteins 64N and 96N,
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respectively (Figure S2e). The resulting SI-Bricks parts/vectors allow for easy swapping of any
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IntC, multimeric material protein, or IntN of interest by digestion with EcoRI/KpnI, KpnI/Kpn2I,
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or Kpn2I/XhoI, respectively. Additionally, the presence of NheI/SpeI sites allows for in situ
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recursive directional genetic assembly of any large, repetitive material proteins of interest
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(Supplementary Note 1, Figure S1, Figure S2). Upregulation of GlyV tRNA production. In addition to sequence optimizations, cellular
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glycyl
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spidroin overexpression. The glyV tRNA coding sequence and its native promoter were PCR-
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amplified from the NEB10β genomic DNA (Table S2) and cloned between AatII/XhoI sites of a
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medium copy vector carrying p15A replication origin and Kanamycin resistance (KanR), yielding
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plasmid pGlyV (Table S3). For all spidroin expression, pGlyV was co-transformed with the
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spidroin plasmid.
tRNA levels were also upregulated to meet the high demands on
glycyl
tRNA posed by
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Shake flask cultures. For initial ligation tests (Figure S3), protein production was carried
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out in shake flasks. Transformants were cultured overnight in 50 mL TB medium at 37 °C on an
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orbital shaker. Overnight 50 mL cultures were then used to inoculate 500 mL fresh TB medium
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in Erlenmeyer flasks at an initial OD600 = 0.08. Cultures were grown at 37 °C with orbital
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shaking to OD600 = 6, then induced by addition of 1 mM IPTG and cultured for an additional 6
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hours at 30 °C with orbital shaking.
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Bioproduction in fed-batch bioreactors. All spidroins were ultimately produced in 2 L
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fed-batch bioreactors (Bioflo120, Eppendorf). Transformants were cultured overnight in 50 mL
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TB medium at 37 °C on an orbital shaker. The overnight cultures were then used to inoculate 1 L
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glucose M9 medium with tryptone supplement at an initial OD600 = 0.08. After overnight
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incubation at 37 °C with orbital shaking, 1 L cultures were pelleted by centrifugation at 4500 x g
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for 10 min and resuspended in 300 mL sterile resuspension medium (250 mM MOPS pH 7.4,
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2.5% w/v glucose, 60 g/L tryptone, 25 mM sodium citrate, 10 mM MgSO4, 500 µM
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FeSO4·7H2O, 15 µM thiamine, 5x micronutrients). The resuspended cultures were then added to
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an autoclaved 2 L Bioflo120 heat-blanketed bioreactor containing 1.2 L water and 1.15x M9
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salts. Sterile CaCl2·2H2O was added to a final concentration of 100 µM. Antifoam 204 was
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added as needed to minimize foaming (approximately 0.01% v/v). Agitation and air flow was
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regulated to maintain approximately 70% dissolved oxygen (DO). After consumption of the
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initial 0.5% w/v glucose (as judged by ∆DO), a sterile substrate feed (20% w/v glucose, 48 g/L
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tryptone, and 10 g/L MgSO4·7H2O) was initiated to maintain a linear growth rate. Reactors were
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induced at OD600 = 80 by addition of 1 mM IPTG and culture temperature was reduced to 30 °C.
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Cultures were collected four hours after induction. Based on densitometric analysis of
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Coomassie Blue-stained SDS-PAGE gels (Figure S4), final product titer for 96-mer reactants
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was estimated at approximately 2 g/L or 63 mg/g cell dry weight.
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Protein ligation. Cell cultures were pelleted by centrifugation at 4500 x g for 30 min.
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Pellets from complimentary SI-fused spidroins (e.g. 96N and C96 or 64N and C64) were combined
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at a 1:1 reactant ratio based on densitometric analysis of Coomassie Blue-stained SDS-PAGE
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gels. Mixed pellets were resuspended in sonication buffer (300 mM NaCl, 20 mM MOPS pH
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7.4, 2 mM TCEP, 1 mM PMSF) and sonicated using a QSonica Q700 sonicator (Qsonica) for 10
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min (5s on, 10s off). Sonicated resuspensions were pelleted by centrifugation at 25,000 x g for
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30 min to remove supernatants. Pellets were resuspended in ligation buffer (8 M urea, 20 mM
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MOPS pH 7.4, 300 mM NaCl, and 2 mM TCEP) and stirred at 37 °C for 24 h to dissolve SI-
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fused spidroins and ensure complete ligation. The mixtures were then centrifuged at 25,000 x g
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for 1 h to remove cell debris and undissolved proteins.
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Protein purification. The purification protocol was modified from previous methods.9
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Specifically, ligated spidroins in ligation buffer were acidified to pH 4.0 with acetic acid.
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Ammonium sulfate was then added to a final concentration of 1.2 M. The mixture was then
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centrifuged at 40,000 x g for 30 min. The pellet was discarded, and additional ammonium sulfate
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was added to the supernatant to a final concentration of 2.3 M. After stirring for 1 h, the mixture
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was centrifuged again at 40,000 x g for 15 min., yielding a pellet containing approximately 75%
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spidroin as estimated by Coomassie Blue-stained SDS-PAGE gels. The supernatant was
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discarded, and the pellet was resuspended in SEC buffer (8 M urea, 10 mM ammonium
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bicarbonate pH 10) for further purification by size-exclusion chromatography. SEC purifications
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were performed on an AKTA Pure Chromatography System (GE Healthcare Life Sciences)
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using a HiPrep 16/60 Sephacryl S-500 HR column (for 128-mer and 192-mer) or a HiPrep 16/60
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Sephacryl S-400 HR column (for 96-mer). Proteins were separated using an isocratic elution
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with SEC buffer at a flow rate of 0.5 mL/min. Fractions containing greater than 90% ligation
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product, as determined by SDS-PAGE gel densitometry, were collected, resulting in
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approximately 5% final product recovery as judged by product dry mass. SEC-purified fractions
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were combined and dialyzed in 10K MWCO SnakeSkin dialysis tubing (ThermoFisher
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Scientific), followed by lyophilization.
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Ligation kinetics analysis. For kinetics analysis, 64-mer protein concentrations in crude
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lysates were estimated by densitometric analysis of Coomassie Blue-stained SDS-PAGE gels.
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Based on estimated concentrations, fully sonicated resuspensions of 64C and N64 in ligation
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buffer were combined to give final concentrations of 100 µM for both 64C and N64 in a final
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volume of 500 µL. These mixtures were pelleted by centrifugation, and pellets were resuspended
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in 500 µL of desired test buffer pre-incubated at the desired test temperature. Reactions were
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quenched by transferring 5 µL of reaction to 95 µL of Laemmli sample buffer preheated to 100
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°C and continuing boiling for 10 min.
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SDS-PAGE and densitometric analysis. All SDS-PAGE gels were 1 mm thick,
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discontinuous with 3% stacking gel, and hand cast at the indicated percentages. Samples were
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prepared at 1 mg/mL or 5 µM total protein in Laemmli sample buffer (2% SDS, 10% glycerol,
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60 mM Tris pH 6.8, 0.01% bromophenol blue, 100 µM DTT). Gels were run on Mini-
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PROTEAN Tetra Cells (Bio-Rad) in 1x Tris-glycine SDS buffer (25 mM Tris base, 250 mM
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glycine, 0.1% w/v SDS), until just before the dye front exited the gel. Gels were stained in
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Coomassie Blue solution (50% methanol, 10% w/v acetic acid, 1 g/L Coomassie Brilliant Blue)
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for a minimum of one hour at room temperature with gentle agitation and destained in
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Coomassie Blue destain buffer (40% v/v methanol, 10% v/v acetic acid) for a minimum of one
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hour. Gels were imaged on an Azure c600 Imager (Azure Biosystems). All densitometry analysis
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was performed with the AzureSpot Analysis Software (Azure Biosystems). Images were
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background subtracted with a built-in automatic lane edge subtraction algorithm. Protein band
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intensities were integrated by the AzureSpot software. Ligation yield was calculated as the
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integrated intensity of the product band over the sum of both reactant and product band areas.
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Purity was calculated as the integrated intensity of the product band over the integrated intensity
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of all other bands. Spidroin titer was calculated as the integrated intensity of the spidroin band
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over the integrated intensity of all other bands multiplied by the measured cell density (OD600) of
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culture at the time of sampling and 150 mg/L/OD600 (150 mg/L/OD600 is an average typical total
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protein titer for E. coli grown in glucose minimal medium).20
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Fiber spinning and mechanical testing. Fiber spinning and mechanical testing were
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performed following a protocol modified from previous methods.9,21,22 Lyophilized spidroin
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powders were dissolved in hexafluorisopropanol (HFIP) to 17% w/v. This protein dope was
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loaded to a 100 µL Hamilton gastight syringe (Hamilton Robotics) fitted with a 23s gauge (116
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µm inner diameter, 1.71 inch length) needle. The syringe was fitted to a Harvard Apparatus
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Pump 11 Elite syringe pump (Harvard Apparatus), and the dope was extruded into a 95% v/v
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methanol bath at 5 µL/min. Extruded fibers were then transferred to a 75% v/v methanol bath
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and carefully extended at approximately 1 cm/s to the maximum draw ratio (~600%) without
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fiber fracture. Extended fibers were removed from the bath and held under tension until visibly
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dry. Segments of post-drawn fibers (20 mm) were carefully laid exactly vertical across a 5 mm
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(vertical) x 15 mm (horizontal) opening cut into a 20 mm x 20 mm piece of cardstock and fixed
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with adhesive tape at both ends of the opening. Diameters of mounted fibers were then measured
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by light microscopy, averaging measurements at three points along the fiber axis (Tables S4-S6,
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Figure S5a). Mechanical properties were measured by axial pull tests on an MTS Criterion
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Model 41 universal test frame fitted with a 1 N load cell (MTS Systems Corporation). Cardstock
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holders were mounted between two opposing spring-loaded grips, and the supporting edges were
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carefully cut. Pull tests were conducted at a relative humidity of 30% and temperature of 22 °C,
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with a constant crosshead speed of 10 mm/min. Stress-strain curves were recorded by the MTS
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TW Elite test suite at a sampling rate of 50 Hz. Fiber breaks were recorded when a 90% drop
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from peak stress was detected. All mechanical properties were automatically calculated by the
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MTS TW Elite test suite. Ultimate tensile strength was calculated as the maximum measured
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load over the initial fiber cross-sectional area ( ݎߨ = ܣଶ ), as determined from measured initial
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diameters (see Supplementary Note 2 for an analysis of the validity of this approach). Modulus
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was calculated as the slope of a linear least squares fit to the stress/strain data of the initial elastic
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region. Toughness was calculated as the area under the total stress/strain curve divided by the
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initial fiber volume (ܸ = ߨ ݎଶ ℎ) as calculated from measured initial fiber diameters and set initial
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gage length of 5 mm. For each protein, a total of 14 fibers were measured in this manner.
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Light microscopy. Fiber diameters were measured using images acquired with a Zeiss
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Axio Observer ZI Inverted Microscope equipped with a phase contrast 20x objective lens and the
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Axiovision LE software (Zeiss). For morphological analysis and further confirmation of fiber
275
diameters, additional images were acquired with a Nikon Eclipse TiE Inverted Microscope
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equipped with a 60x objective and analyzed using the Nis-Elements software (Nikon; Figure
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S5a).
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Scanning Electron Microscopy. Following tensile tests, silk fibers were mounted onto a
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sample holder using conductive tape. The sample holder was sputter coated with a 10 nm gold
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layer using a Leica EM ACE600 high vacuum sputter coater (Leica Microsystems). Fibers were
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imaged using a Nova NanoSEM 230 Field Emission Scanning Electron Microscope (Field
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Electron and Ion Company, FEI) at an accelerating voltage of 7-10 kV. Fiber surface roughness
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(both exterior and interior) was calculated using MountainsMap SEM Topo software (Digital
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Surf). The root mean square height (Sq) of each surface was determined from single images
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using greyscale values, normalized by the entire range of values in each micrograph. To remove
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fiber curvature from consideration, a standard Gaussian filter with a threshold of 2.5 µm was
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applied when analyzing exterior surfaces. For convenience, exterior and interior surface
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roughness values are presented relative to each other, with the highest interior value set at 1.
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Fiber circularity was calculated from cross-sectional areas and perimeters as 4ߨ(௧ మ ),
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where a perfect circle gives a value of 1.
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Polarized Raman spectromicroscopy. The method reported here is adapted from
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several previous studies of molecular alignment in spider silk fibers.23,24,25 Silk fibers were
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carefully fixed to glass microscope slides with microscale markings to ensure that spectra were
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acquired at the same location before and after stage rotation. Raman spectra were acquired with a
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Renishaw RM1000 InVia Confocal Raman Spectrometer (Renishaw) coupled to a Leica DM LM
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microscope with rotating stage (Leica Microsystems). Silk fibers were initially oriented along the
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x-axis. Fibers were irradiated at a fixed point with the 514 nm line of an argon laser with
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polarization fixed along the x-axis and focused through a 50x objective (NA = 0.75). Spectra
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were recorded from 1150-1750 cm-1 with an 1800 lines/mm grating. For each acquisition, a total
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of 16 spectra were accumulated, each for 10 s. The stage was then rotated to orient fibers along
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the y-axis with the same laser polarization, and spectra were acquired a second time at the same
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fixed point. No signs of thermal degradation were apparent either visually or within recorded
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spectra. All recorded spectra were analyzed using Fityk 0.9.8.26 Baselines were subtracted from
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all spectra using the built-in Fityk automatic convex hull algorithm. For intensity ratio
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calculations, all spectra were normalized to the intensity of the 1450 cm-1 peak, which arises
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from CH2 bending and is insensitive to protein conformation.24 For each fiber, the normalized
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intensity of the peak at 1670 cm-1 when oriented along the Y-axis was divided by the normalized
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Biomacromolecules
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intensity of the peak when oriented along the X-axis to give the intensity ratio ( ܫ). This
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procedure was performed on a total of three separate fibers and calculated intensity ratios were
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averaged. Spectra were also averaged and presented in Figure 3d with standard deviations for
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each point along the spectra.
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Fourier Transform Infrared Spectroscopy. For secondary structure determination,
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FTIR spectra were acquired with a Thermo Nicolet 470 FT-IR spectrometer (ThermoFisher
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Scientific) fitted with a Smart Performer ATR accessory with Ge crystal. Spectra were acquired
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from 1350-1750 cm-1 at 4 cm-1 resolution. A total of 254 scans were accumulated for each
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sample. All recorded spectra were analyzed using Fityk 0.9.8. Baselines were subtracted from all
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spectra using the built-in Fityk convex hull algorithm. The amide I band (1600 - 1700 cm-1) was
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deconvolved into a set of five Lorentzian peaks centered at 1626.5, 1646.5, 1659, 1679.5, and
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1700 cm-1 for β-sheet, random coil, α-helix, β-turn, and β-sheet components, respectively.
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Component peak assignments were based on those used in previous studies.27 Peak areas were
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integrated and percentages were calculated as the component peak area over the sum of all peak
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areas. Percentages were averaged from measurements of three fibers.
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Statistical analysis. GraphPad Prism 7 (GraphPad Software) was used for statistical data
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analysis, using both two-tailed unpaired t-tests (26 degrees of freedom) and one-way ANOVA
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tests (41 degrees of freedom) to compare data sets.
326 327
RESULTS AND DISCUSSION:
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To facilitate microbial production of highly repetitive, SI-fused material proteins, we
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developed a standardized DNA part assembly system termed SI-Bricks (Supplementary Note 1,
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Figure S1). SI-Bricks allows for rapid genetic swapping of the core components of the
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envisioned SI-mediated ligation platform (i.e. N-inteins, material proteins, C-inteins, and fusion
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domains/purification tags) in addition to common standardized biological parts (e.g. promoters,
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ribosomal binding sites, replication origins, and selection markers) (Table S7), all by means of
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restriction enzyme digestion and enzymatic ligation. To construct the SI-Bricks parts necessary
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for this study, we first assembled 64-mer and 96-mer spidroin DNA sequences by recursive
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directional genetic assembly of a single, codon-optimized repeat unit (1-mer) of the N. clavipes
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dragline spidroin MaSp1.5 The assembled spidroin sequences, flanked by SI-Bricks restriction
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sites, were genetically combined with codon-optimized SI DNA sequences and other necessary
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expression parts (Figure S2). Given the tendency of high MW spidroins to form inclusion bodies
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in microbial hosts, we employed a recently engineered SI pair (Cfa) that retains catalytic activity
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in the presence of 8 M urea,15 a denaturant often used to extract and solubilize spidroins from E.
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coli. The resulting SI-fused spidroins (64N, C64, 96N, and C96) were individually expressed in an
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E. coli host with
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glycine codons in the spidroin sequences. Using our optimized fermentation conditions, typical
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titers of the SI-fused spidroins were approximately 2 g/L from a glucose minimal medium with
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tryptone supplementation after four hours of production (Figure S4).
glycyl
tRNA levels engineered to meet the demands of the most frequently used
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Spidroin inter- and intramolecular interactions can be highly sensitive to salt and pH,
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even in the presence of 8 M urea28,29 and we expected that such interactions would lower ligation
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efficiency of the Cfa SIs. Thus, to test optimum conditions for the ligation of Cfa-fused
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spidroins, 8 M urea extracts of E. coli expressing 64N or C64 were mixed at several salt
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concentrations, temperatures, and pH values (Figure S3). Under all tested conditions, SI-
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mediated spidroin ligation was both rapid and robust, with the highest ligation yields observed at
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37 °C, 300 mM NaCl, pH 7. Thus, for all subsequent ligations, these conditions were maintained,
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giving ligation yields of 68% and 62% for 128-mer and 192-mer spidroins, respectively (Figure
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2a,b).
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Ligation products were initially separated from most cellular proteins by selective
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ammonium sulfate precipitation and then further separated from unreacted 64-mer or 96-mer by
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size exclusion chromatography (SEC) for a final product purity ≥90% (Figure S6). As a standard
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for mechanical properties, a 96-mer spidroin with no SIs was also expressed and purified
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following identical procedures. Spinning dope was prepared from lyophilized powder of each
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purified protein and fibers were spun and mechanically tested following well-documented wet-
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spinning protocols detailed in the Materials and Methods section.
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Comparing post-drawn 96-mer and 192-mer fibers, mechanical testing revealed
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significant, nearly two-fold increases in both tensile strength (from 525 to 1031 MPa, P