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Degenerate Oligonucleotide Gene Shuffling 2032. Stemmer, W. P. C. (1994) Rapid evolution of a protein in vitro by DNA shuffling.Nature 370, 389–391.3. Stemmer, W. P. C. (1994) DNA shuffling by random fragmentation and reassembly:in vitro recombination for molecular evolution. Proc. Natl. Acad. USA 91,10,747–10,751.4. Schmidt-Dannert, C., Umeno, D., and Arnold, F. H. (2000) Molecular breeding ofcarotenoid biosynthetic pathways. Nature Biotechnol. 18, 750–753.5. Kikuchi, M., Ohnishi, K., and Harayama, S. (1999) Novel family shuffling methodsfor the in vitro evolution of enzymes. Gene 236, 159–167.6. Morris, D. D., Gibbs, D. D., Chin, C. W., et al. (1998) Cloning of the xynB genefrom Dictyoglomus thermophilum strain Rt46B.1 and action of the gene-producton kraft pulp. Appl. Environ. Microbiol. 64, 1759–1765.7. Gibbs, M. D., Nevalainen, K. M. H., and Bergquist, P. L. (2001) Degenerateoligonucleotide gene shuffling (DOGS): a method for enhancing the frequency ofrecombination with family shuffling. Gene 271, 13–20.8. Sakka, K., Kojima, Y., Kondo, T., Karita, S., Ohmiya, K., and Shimada, K. (1993)Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanaseA: identification of catalytic and cellulose binding domains. Biosci. Biotechnol.Biochem. 57, 273–277.9. Yang, V. W., Zhuang, Z., Elegir, G., and Jeffries, T. W. (1995) Alkaline-activexylanase produced by an alkaliphilic Bacillus sp isolated from kraft pulp. J. Ind.Microbiol. 15, 434–441.10. Morris, D. D., Gibbs, M. D., Ford, M., Thomas, J., and Bergquist, P. L. (1999) Family10 and 11 xylanase genes from Caldicellulosiruptor isolate Rt69B.1. Extremophiles3, 103–111.11. Fernandes, A. C., Fontes, C. M., Gilbert, H. J., Hazlewood, G. P., Fernandes, T. H.,and Ferreira, L. M. (1999) Homologous xylanases from Clostridium thermocellum:evidence for bi-functional activity, synergism between xylanase catalytic modulesand the presence of xylan-binding domains in enzyme complexes. Biochem. J. 342,105–110.12. Elegir, G., Szakacs, G., and Jeffries, T. W. (1994) Purification, characterization andsubstrate specificity of multiple xylanases from Streptomyces sp strain B-12-2.Appl. Environ. Microbiol. 60, 2609–2615.13. Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., McCallum, C. M., andHenikoff, S. (1998) Consensus-degenerate hybrid oligonucleotide primers foramplification of distantly related sequences. Nucleic Acids Res. 26, 1628–1635.14. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G.(1997) The ClustalX windows interface: flexible strategies for multiple sequencealignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876–4882.15. Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: The European MolecularBiology Open Software Suite. Trends Genet. 16, 276–277.16. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Sitedirectedmutagenesis by overlap extension using the polymerase chain reaction. Gene77, 51–59.
204 Bergquist and Gibbs17. Teather, R. M. and Wood, P. J. (1982) Use of Congo Red polysaccharide interactionin enumeration and characterisation of cellulolytic bacteria from bovinerumen. Appl. Environ. Microbiol. 43, 777–780.18. Lever, M. (1973) Colorimetric and fluorometric carbohydrate determination withp-hydroxybenzoic acid hydrazide. Biochem. Med. 7, 274–281.19. Britton, H. T. S. and Robinson, R. A. (1931) Universal buffer solutions and the dissociationconstant of veronal. J. Chem. Soc. 1, 1456–1462.
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METHODS IN MOLECULAR BIOLOGY 352Pr
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M E T H O D S I N M O L E C U L A R
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PrefaceProtein engineering is a fas
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ContentsPreface ...................
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ContributorsKATJA M. ARNDT • Inst
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Contributors xiKAZUNARI TAIRA • D
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1Combinatorial Protein Design Strat
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Combinatorial Protein Design Strate
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2Global Incorporation of Unnatural
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Incorporation of Unnatural Amino Ac
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3Considerations in the Design and O
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Design of Coiled Coil Structures 37
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4Calcium Indicators Based on Calmod
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Protein-Based Ca 2+ Indicators 73Fi
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Protein-Based Ca 2+ Indicators 7512
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Protein-Based Ca 2+ Indicators 8145
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5Design and Synthesis of Artificial
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Design of Zinc Finger Proteins 853.
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Design of Zinc Finger Proteins 89pr
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Design of Zinc Finger Proteins 91Fi
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96 Koide and Koidewhile retaining t
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98 Koide and Koide5. M9-tryptone: M
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100 Koide and Koidetarget-binding s
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102 Koide and Koide4. Discard the s
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104 Koide and Koideup to 1 mM for h
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106 Koide and Koideplate. Incubate
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108 Koide and Koide1. Perform steps
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7Engineering Site-Specific Endonucl
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Engineering Site-Specific Endonucle
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115Fig. 1. Mapping group-specific r
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8Protein Library Design and Screeni
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Protein Library Design and Screenin
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135Fig. 2. Excel worksheet describi
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137Fig. 4. Excel worksheet describi
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Page 166 and 167: Protein Library Design and Screenin
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Page 170 and 171: Protein Design by Binary Patterning
Page 180 and 181: 10Versatile DNA Fragmentation and D
Page 182 and 183: NExT DNA Shuffling 169This chapter
Page 184 and 185: NExT DNA Shuffling 1713. Methods3.1
Page 186 and 187: NExT DNA Shuffling 17372°C, 4 min.
Page 188 and 189: NExT DNA Shuffling 175Fig. 2. Varia
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Page 192 and 193: NExT DNA Shuffling 179Fig. 3. Reass
Page 194 and 195: 181
Page 196 and 197: NExT DNA Shuffling 183likelihood of
Page 198 and 199: NExT DNA Shuffling 1853.9.2. Calibr
Page 200 and 201: NExT DNA Shuffling 187staining. Bec
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Page 204 and 205: 11Degenerate Oligonucleotide Gene S
Page 206 and 207: Degenerate Oligonucleotide Gene Shu
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Page 220 and 221: Engineered M13 Bacteriophage Coat P
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Page 235 and 236: 222 Fujita et al.The methods that h
Page 237 and 238: 224 Fujita et al.3. Streptavidin an
Page 239 and 240: 226 Fujita et al.Fig. 2. (Continued
Page 241 and 242: 228 Fujita et al.Fig. 3. (Continued
Page 243 and 244: 230 Fujita et al.Fig. 3. (A) Schema
Page 245 and 246: 232 Fujita et al.1. Add 2 µg mRNA
Page 247 and 248: 234 Fujita et al.Innovative Bioscie
Page 249 and 250: 236 Fujita et al.30. Kim, Y., Mlsa,
Page 251 and 252: 238 Ghadessy and Holligeraffinity m
Page 253 and 254: 240 Ghadessy and HolligerFig. 1. (A
Page 255 and 256: 242 Ghadessy and HolligerFinally, t
Page 257 and 258: 244 Ghadessy and Holliger2. Prepare
Page 259 and 260: 246 Ghadessy and Holliger2. After 6
Page 261 and 262: 248 Ghadessy and Holliger21. Oberho
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254 Campbell-Valois and Michnick7.
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256 Campbell-Valois and MichnickFig
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258 Campbell-Valois and Michnickres
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260Fig. 3. BL21 pREP4 cells were co
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262 Campbell-Valois and MichnickFig
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264 Campbell-Valois and Michnickvar
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276 Hecky et al.improved half-life
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278 Hecky et al.Fig. 1. (A) Scheme
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280 Hecky et al.11. Reverse primer
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282 Hecky et al.high variability in
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284 Hecky et al.coding sequence for
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286 Hecky et al.4. Analyze the resu
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Table 1Amino Acid Substitutions Sel
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290 Hecky et al.Fig. 4. Structure o
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292 Hecky et al.Fig. 5. Expression
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294 Hecky et al.Table 2Kinetic Para
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296 Hecky et al.The thermoactivity
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298 Hecky et al.Fig. 8. Unfolding o
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300 Hecky et al.for the first trans
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302 Hecky et al.7. Thompson, M. J.
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304 Hecky et al.40. Wang, X., Minas
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306 IndexCCalcium indicators, see C
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308 Indexchemiocompetent cellprepar
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310 Indexmaterials, 169, 170mutatio
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312 IndexTerminal truncation, see a
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