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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

DOI:
10.3791/2505-v

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09:01 min

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March 16, 2011

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, , , ,
1Department of Microbiology & Environmental Toxicology,University of California Santa Cruz – UCSC

Chapters

  • 00:05Title
  • 01:38Creation of a Mutant Library: Mutagenesis
  • 03:23Creation of a Mutant Library: Readout
  • 04:03Mutant Identification and Characterization: Construction of Gradient
  • 05:06Mutant Identification and Characterization: Stamp Transfer of Bacteria
  • 06:27Mutagenesis and Functional Selection Protocols: Representative Results
  • 08:14Conclusion

Summary

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Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Tags

MutagenesisFunctional SelectionDirected EvolutionProteinsE. ColiMutant LibrariesColE1 PlasmidDNA Polymerase IMutator StrainSolid MediaMutation FrequencyLabelingPCRCloningSequence Space ExplorationBiochemical Activities

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