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1 4 Alternative methods for library generation There are a few methods for making a library that don't fit into either category previously discussed. We will read about two of these methods: chemical mutagenesis and the use of mutator strains. We will discuss why these methods are not as general or as widely used as those discussed previously. Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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2 13 Alternative methods for enzyme/catalyst design We will wrap up our discussion about directed evolution and compare and contrast the methods we have learned about in previous weeks. This week we will also spend time looking at approaches for enzyme evolution based on rational design and discuss the advantages or disadvantages of these methods as compared with directed evolution. Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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3 12 Enzyme evolution by in vitro compartmentalization In vitro compartmentalization (IVC) is based on water-in-oil emulsions where surfactants aid in the formation of microscopic aqueous compartments. These compartments simulate cellular compartments in which only the reaction for selection is performed. Inside each droplet transcription, translation, and expression of the desired protein takes place using active components. This method is poised to become the most promising for the future of enzyme evolution – we'll discuss why. Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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4 3 Library generation by recombination We will continue our discussion of library generation examining methods that are based on recombination, meaning DNA is broken and rejoined in new combinations. DNA shuffling, the best known method of recombination, allows useful mutations from multiple genes to be combined. Many methods have been developed based on this principle. Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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5 8 Enzyme evolution using phage display (cont.) Affinity selection for improved catalysts has limitations; not all enzymatic activities have suitable suicide substrates. Direct selection of enzymes on the basis of catalysis is possible using phage display technology, but is more complex as a physical link between the phage-bound enzyme and the substrate must be established. We will discuss how this type of selection is achieved using two different examples of substrate linkage Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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6 5 Enzyme evolution by genetic complementation In enzyme evolution, it is important to link the gene of interest, the enzyme it encodes and the product of the activity of the enzyme. Many methods exist for the selection of particular library members based on enzyme activity. We will begin be examining in vivo selection systems, which link cell survival to enzyme activity. Genetic complementation, the most basic of these selection systems, is the rescuing of a specific genetic defect leading to a particular phenotype by the addition of DNA encoding a gene of interest. Genetic complementation is often used to investigate the function of genes for which no function has previously been described. Notes [开放课件]

授课人:Dr. Kerry Love

课程:Directed Evolution: Engineering Biocatalysts,

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使用许可:CC BY-NC-SA

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