Chemical and Biosynthetic MethodsToward Mimicking Nature’s Strong Fiber:
Spider Dragline Silk
Maren E. Buck
Lynn Group
5/3/2007
2
Outline
I. Introduction to spider silks and silk structure
II. Biosynthetic methods to produce silk protein analogs
III. Chemical methods to synthesize silk-like polymers
IV. Applications
V. Conclusions
3Vollrath, F. J. Biotechnol. 2000, 74, 67-83.Hu, X. et al. Cell. Mol. Life Sci. 2006, 63, 1986-1999.
Spiders spin 6 different fibers
Web reinforcement (Minor ampullate 1 and 2) Dragline (major
ampullate 1 and 2)
Wrapping and egg case fiber (aciniform)
Pyriform silk (?)
Acini-form
Capture Spiral(Flagelliform)
Glue coating(Aggregate silk) (?)
Large diameter eggCase fiber (Tubuliform)
Aggregate TubuliformFlagelliform
Pyriform
Minor ampullate
Major ampullate
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The classic strong synthetic fiber
Material Strength (GPa) Elasticity (%) Energy to break (J/kg)
Dragline Silk 1.1 35 4 x 105
Kevlar 3.6 5 3 x 104
Rubber 0.001 600 8 x 104
Nylon, type 6 0.07 200 6 x 104
Fiber axis
Kevlar®: Dupont (1960s) Uses
- Bulletproof vests and helmets- Automobile brake pads- Ropes and cables- Aerospace components
Lewis, R. Chem. Rev. 2006, 106, 3762-3774. Vollrath, F.; Knight, D.P. Nature 2001, 410, 541-548.Tanner, D.; Fitzgerald, J.A.; Phillips, B.R. Angew. Chem. Int. Ed. Engl. Adv. Mater. 1989, 5, 649-654.Kubik, S. Angew. Chem. Int. Ed. 2002, 41, 2721-2723.
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Spider silks have potential in many applications
Surgical sutures Scaffolds for tissue engineering
Biomedical applications
Parachutes
High strength ropes/cables
Fishing line
Technical and industrial applications
Ballistics
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Forced silking to obtain silk fibers
Spiders are anesthetized with CO2
and secured ventral side up
Silk is pulled from the spinneret,
attached to a reel, and drawn at a
specified speed
Work, R. W.; Emerson, P. D. J. Arachnol. 1982, 10, 1-10.Elices, M.; Perez-Rigueiro, J.; Plaza, G. R.; Guinea, G. V. JOM 2005, 57.
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Spiders are highly developed fiber “spinners”
Lewis, R. Chem. Rev. 2006, 106, 3762-3774.Dicko, C.; Vollrath, F.; Kenney, J.M. Biomacromolecules 2004, 5, 704-710.
Spidroin secretion
Lumen
Spinneret
Duct
Fiber alignment
Duct
Tail
Funnel
1 mm
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Primary structure of spider dragline silk
Hinman, M.B.; Jones, J. A.; Lewis, R. TIBTECH 2000, 18, 374-379. Vollrath, F.; Knight, D. P. Nature 2001, 410, 541-548.Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87.
QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGAGQGGRGAGQS
SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGQGGRGAGQNSQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG
GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQSSQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG
GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQSSQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG
GLGGYGRQGAGGAAAAAAGAGQGGRGAGQSNQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG
GLGGYGGQGAGGAAAAAGQGGRGAGQNSQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG
GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQSSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG
SSLRSAAAAASAASAGS
Fibrous protein composed of Spidroin 1 (MaSp1) and Spidroin 2 (MaSp2)- Sequences highly conserved- Repetitive stretches of poly(Ala) and (GlyGlyXaa)n sequences (Xaa = Tyr, Leu, Gln)- MW of MaSp1 ~ 275-320 kDa; Sp1+Sp2 ~ 700-750 kDa
Repeating sequence of MaSp1
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Antiparallel and parallel -sheet structure
Poly(alanine) segment
Rotondi, K. S.; Gierasch, L. M. Biopolymers 2005, 84, 13-22. Simmons, A.; Ray, E.; Jelinski, L. W. Macromolecules 1994, 27, 5235-5237.
N-terminus
N-terminus
C-terminus
C-terminus
N-terminus
N-terminus
C-terminus
C-terminus
N C
NC
N C
N C
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Solid state 13C-NMR and FT-IR spectroscopy
Marcotte, I.; van Beek, J. D.; Meier, B. H. Macromolecules 2007, 40, 1995-2001.Simmons, A.; Ray, E.; Jelinski, L.W. Macromolecules 1994, 27, 5235-5237.Dong, Z.; Lewis, R.; Middaugh, C. R. Arch. Biochem. Biophys. 1991, 1, 53-57.
13C-NMR chemical shifts (ppm)
13C-labeledAlanine
Wavenumber (cm-1)
1700 1600 15000.1550
0.2800
0.4050
Ab
sorb
an
ce
1691
1666
1637
1612
Infrared spectrum of silk from Nephila clavipes
Amide I (antiparallel-sheet)
-carbon
-carbon
Anti-parallel β-sheet
Parallel β-sheet
α-helixAla C
α-helix
Ala C
Ala CC=O
-sheet
20.1 15.1
48.7 52.5
171.9 176.5
Infrared wavelengths (cm-1)
1630, 1685
1630, 1645
1650, 1560
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Proposed secondary structure and mode of elasticity
Kubik, S. Angew. Chem. Int. Ed. 2002, 41, 2721-2723.Van Beek, J. D.; Hess, S.; Vollrath, F. Meier, B. H. Proc. Nat. Acad. Sci. 2002, 99, 10266-10271.
• Poly(Ala) modules form anti-parallel β-sheets (~30-40%)• Glycine-rich, amorphous regions are thought to be helical
Disordered chain region
Strain
Crystalline region with-sheet structure
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Synthetic approaches to spider dragline silk
Biosynthesis Chemical Synthesis
Protein sequences
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Two biosynthetic routes to spidroin proteins
Vendrely, C.; Scheibel, T. Macromol. Biosci. 2007, 7, 401-409.Altman, G.H. et al. Biomaterials 2003, 24, 401-416.
Synthetic DNA
Spider cDNA
Flexibility withhost
Protein fibers
Reverse transcription
Eukaryotic host (insect cells)
Spider silk protein sequences/mRNA
Gene design
Nephila clavipes
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Expression of spider silk cDNA in mammalian cells
Lazaris, A. et al. Science 2002, 295, 472-476.
Dragline silk gene sequencefrom A. diadematus
Gene sequence inserted intoexpression vector
Transformation of vector in mammalian cells
protein synthesis
Protein purification, and characterization
Protein: MW ~ 60-140 kDa Fiber diameter ~ 40 μm Yield ~ 37 mg/L
Mechanical Properties:
Protein sample Toughness(MJ/m3)
Modulus(GPa)
Elasticity(%)
Strength(GPa)
ADF-3
A. diadematus dragline
85 13 43.4 0.26
130 10 30 1.1
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Recombinant expression of synthetic silk genes
DNA fragment
Fahnestock, S. R.; Irwin, S. L. Appl. Microbiol. Biotechnol. 1997, 47, 23-32. Stephens, S.J. et al. Mat. Res. Soc. Symp. Proc. 2003, 774, 2.3.1-2.3.10.Fahnestock, S. R.; Bedzyk, L. A. Appl. Microbiol.Biotechnol. 1997, 47, 33-39.O’Brien, J. P.; Fahnestock, S. R.; Termonia, Y.; Gardner, K. H. Adv. Mater. 1998, 10, 1185-1195.
AGQGGYGGLGSQG--------------------------------------------AGQGGYGGLGSQGAGRGGLGGQGAGAAAAAAAGGAGQG-------GLGSQGA---------- GQGAGAAAAAA----GGAGQGGYGGLGSQGAGRG-----GQGAGAAAAAA---GG
Spidroin 1 analog: DP-1B[
]n=8-16
Ligate 8 or 16DNA fragments
DNA duplex
Hybridize complementary
strands
Premature termination with expression in E. coli
High MW polymers from yeast
Transform inEscherichia coli
Insert gene into plasmid vector
Or transform inyeast
Protein fibers1 g/L
Protein fibers300 mg/L
170 nm diameter fibers
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Summary of biosynthetic pathways
Biosynthetic Method Advantages Disadvantages
Spider Silk cDNA Difficulty with protein
purification (aggregation)Produce proteins most
like native silk
High MW polymers
are readily attainable
Eukaryotic hosts are expensive
Synthetic DNA Polymer structure can
be tuned based on
DNA sequence used
Flexibility with
expression host
Truncated syntheses in
many hosts
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Synthetic approaches to spider dragline silk
Biosynthesis Chemical Synthesis
Protein sequences
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Chemical approaches to synthesizing silk-like polymers
Poly(Ala) blocks - PEG linker - Alkyl linkers
Protein structure and properties
Non-peptidepolymers
Living polymerization of peptide monomers
Lego approach(-sheet template) - Rigid or short linkers - Long, flexible linkers
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Synthesis of silk-like polymers: “Lego” approach
Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876.
Linkers -sheet nucleation center Peptide sequence (GAGA)
+
A
B +
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Synthesis of the building blocks
Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876.Wagner, G.; Feigel, M. Tetrahedron 1993, 49, 10831-10842.
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Spectroscopic evidence for the requiredphenoxathiin template
3 4
Flexible linear peptide
Peptides with phenoxathiin template
Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876.
1 2
3424 cm-1
3336 cm-1
3a
3b
4
3407 cm-1
3342 cm-13415 cm-1
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Polymerization of the building blocks
Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876.
Interfacial Polymerization
Solution Polymerization
Monomer A
Monomer B
Copolymer AB
“Nylon Rope Trick”
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Polymerization results
% Yield – Interfacial:
% Yield – Solution:
Mn (Solution) (g/mol):
PDI:
576019,1002.08
505620,6001.79
463917,4001.54
826720,2001.79
Mn = average molecular weight of sample
PDI = distribution of molecular weights in a sample
Winningham, M. J.; Sogah, D. Y. Macromolecules 1997, 30, 862-876.
Spider silk: Mn = ~ 605,000 g/mol (Sp1+Sp2)
PDI = 1.05
P1 P2 P3 P4
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FT-IR characterization of the polymer structure
Winningham, M.J.; Sogah, D.Y. Macromolecules. 1997, 30, 862-876.
1: 2:
Polymer 1 or 2
Peptide 1 or 2
Polymer 1
Peptide 1Peptide 2
Polymer 2
1645 cm-1
1645 cm-1
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Phenoxathiin template with ethylene glycol linkers
57% yieldMn = 22,400PDI=1.72
60% yieldMn = 14,000PDI = 2.4
Interfacial:
Solution:
Rathore, O.; Winningham, M. J.; Sogah, D.Y. J. Polym. Sci: Part A, Polym Chem. 2000, 38, 352-366.Dattagupta, N.; U.S. Patent 4,968,602; 1990.
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13C-NMR spectra suggest -sheet structure
Interfacial polymerization
SolutionpolymerizationTotal -sheet content:
- Interfacial polymerization: 40% - Solution polymerization: 80%
Spider silk -sheet content: 30-40%
Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym Chem. 2000, 38, 352-366.
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Changes in interfacial polymer after annealing above Tg
Solution polymerization
Rathore, O.; Winningham, M. J.; Sogah, D. Y. J. Polym. Sci: Part A, Polym. Chem. 2000, 38, 352-366.
2nd derivative
Raw 1647 cm-1
1628 cm-11635 cm-11683 cm-1
Raw
2nd derivative
1647 cm-1
1633 cm-1
1683 cm-1
Initial Post Annealing
• Polymerization procedure affects structure
• Heating above Tg enhances -sheet content in interfacial polymer
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Chemical approaches to synthesizing silk-like polymers
Poly(Ala) blocks - PEG linker - Alkyl linkers
Protein structure and properties
Non-peptidepolymers
Living polymerization of peptide monomers
Lego approach(-sheet template) - Rigid or short linkers - Long, flexible linkers
Poly(Ala) blocks - PEG linker - Alkyl linkers
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Non-templated polymeric dragline silk mimics
Generic polymer structurepeptide
[
]soft linker
x ~ 4 or 6n ~ 13
QGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLGGQGAGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGAGQGGRGAGQS
SQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLGGLGGYGGQGAGGAAAAAAGQGGRGAGQNSQGAGRGGLGGQAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG
GLGGYGGQGAGGAAAASAGAGQGAGQGGLGGQGAGGAAAAAAAGAGQGGLGGRGAGQSSQGAGRGGEGAGAAAAAAGGAGQGGYGGLGGQGAGQGGYGGLG
GLGGYGGQGAGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAGAGQGGLGGRGAGQSSQGAGRGGLGGQGAGAVAAAAGGAGQGGYGGLG
GLGGYGRQGAGGAAAAAAGAGQGGRGAGQSNQGAGRGGLGGQGAGAAAAAAAGGAGQGGYGGLG
GLGGYGGQGAGGAAAAAGQGGRGAGQNSQGAGRGGQGAGAAAAAAVGAGQEGIRGQGAGQGGYGGLG
GAGGYGGQRVGGAAAAAAGAGQGAGQGGLGGQGAGGAAAAAAGAGQGGLGGRGSGQSSQGAGRGGQGAGAAAAAAGGAGQGGYGGLGGQGVGRGGLGGQGAGAAAAGGAGQGGYGGVG
SSLRSAAAAASAASAGS
Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87.Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.
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Synthesis of triblock copolymers with poly(Ala)
Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.
Water-soluble fraction(46%)
Water-insoluble fraction(54%)
P1: x~4, n~13; 75% yieldP2: x~6, n~13; 69% yield
+
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Polymer
P1 410±35
750±156
Modulus(MPa)
Tensile strength(MPa)
Elongationat break (%)
13.0±1.4
14.2±2.7
22.9±13.6
5.4±1.7P2
Silk (N. clavipes)
Mechanical properties:
22,000 1,100 34
Mechanical properties of the polymer fibers
FT-IR and 13C-NMR indicate formation of anti-parallel -sheets
P1: x~4, n~13P2: x~6, n~13
Rathore, O.; Sogah, D. Y. J. Am. Chem. Soc. 2001, 123, 5231-5239.
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Synthesis of silk-like multiblock copolymers containing flexible alkyl linkers
3 cycles
Yao, J. et al. Macromolecules 2003, 36, 7508-7512.
Yield: 70%MW (viscosity): 44,900
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Multiblock copolymers with poly(isoprene) as the “soft” linker
Zhou, C. et al. Biomacromolecules 2006, 7, 2415-2419.
n = 31 (Mn=2200)n = 72 (Mn=5000)
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13C-NMR and FT-IR characterization of the polymers
Zhou, C. et al. Biomacromolecules 2006, 7, 2415-2419.
P1
Chemical shift (ppm)
P1
P2
P1
Wavenumber (cm-1)
Ab
so
rba
nc
e
1655
1643 1630
176171
52
48
18
P2
P1: n = 31P2: n = 72
35
Chemical approaches to synthesizing silk-like polymers
Poly(Ala) blocks - PEG linker - Alkyl linkers
Protein structure and properties
Non-peptidepolymers
Living polymerization of peptide monomers
Lego approach(-sheet template) - Rigid or short linkers - Long, flexible linkers
Living polymerization of peptide monomers
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Atom transfer radical polymerization (ATRP) of silk-like triblock copolymers
Mn (GPC): 11.5 kDaPDI: 1.29
Mn (GPC): 4.6 kDaPDI: 1.17
Ayres, L. et al. Biomacromolecules 2005, 6, 825-831.
37
Chemical approaches to synthesizing silk-like polymers
Poly(Ala) blocks - PEG linker - Alkyl linkers
Protein structure and properties
Non-peptidepolymers
Living polymerization of peptide monomers
Lego approach(-sheet template) - Rigid or short linkers - Long, flexible linkers
Non-peptidepolymers
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Silk-like polymers without peptide motifs
Soft segment Hard segment
Endcapped macrodiol
Macrodiol
James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer 2006, 47, 3073-3082.
% Hard segment:P1 = 26% P3 = 43%P2 = 33% P4 = 47%
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Mechanical properties of poly(urethane) polymers
Polymer
P1
Tensile strength (MPa)
Elongation at break (%)
587
460
14.9
18.1
72.5
200P2
Modulus (MPa)
447 23.6 156P3
202 18.2 198P4
65.1
59.2
Toughness (MJ/m3)
77.4
31.6
34 1,100 22,000Spider dragline silk 160
James-Korley, L. T.; Pate, B. D.; Thomas, E. L.; Hammond, P. T. Polymer 2006, 47, 3073-3082.Cuniff, P.M. et al. Polym. Adv. Tech. 2003, 5, 401-410.
Soft segment Hard segment
P1 = 26% P3 = 43%P2 = 33% P4 = 47%
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Living polymerization of peptide monomers - Forms -sheets - Control over MW of peptide blocks - Low PDI
Lego approach(-sheet template) - Forms -sheets - Brittle, non-fibrous
Poly(Ala) blocks - Forms -sheets - Produces fibers; not as strong as native silk
Non-peptide polymers - Self-assembles into fibers - High elasticity, low strength
Summary of chemical synthetic pathways
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Applications for spider dragline silk: Tissue Engineering
Allmeling, C.; Jokuszies, A.; Reimers, K.; Kall, S.; Vogt, P. M. J. Cell. Mol. Med. 2006, 10. 1-8.Altman, G. H. et al. Biomaterials 2003, 24, 401-416.
Light micrograph of artificial nerve construct
Artificial nerve grafts:
Artificial ligaments:- Silks promote proliferation of bone marrow cells
- High tensile strengths could restore knee function immediately
- Nerve cells attach and grow on spider silk fibers - Nerve construct composed of pig venules, filled with cells seeded on silk fibers
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Spider silks have potential in many applications
Surgical sutures Scaffolds for tissue engineering
Biomedical applications
Parachutes
High strength ropes/cables
Fishing line
Technical and industrial applications
Ballistics
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BioSteel®
Lazaris, A. et al. Science 2002, 295, 472-476.Karatzas, C. N.; Turcotte, C. 2003, PCT Int. Appl. WO03057727.Karatzas, C. 2001, PCT Int. Appl. WO0156626.Islam, S. et al. 2004, U.S. Pat. 20040102614.
- Genetically modified goats produce silk in mammary glands
- Silk is spun from the goats’ milkExtrusion through “spinnerets” produces fibersAqueous spinning process is environmentally friendly
- Anticipated uses:
Surgical suturesAdhesivesFishing lineBody armor/military applications
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Conclusions
- Spiders can spin fibers with exceptional strength, elasticity, and toughness
- Biosynthetic methods have generated fibers with structure and properties approaching those of native silks
- Chemists can use spider silk as a model to design new fibers and materials with silk-like properties
- Silk-spinning processes must be optimized in order for commercialization to occur
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Acknowledgments
Professor David Lynn
Lynn Group Members:Jingtao ZhangXianghui LiuChris JewellNat FredinBin SunMike KinsingerEric SaurerRyan FlessnerShane Bechler
Practice talk attendees:Lauren BoyleClaire PoppeJulee ByramBecca SplainAlex ClemensKatherine TraynorRichard GrantMatt WindsorMargie Mattmann