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Презентация на тему: Directed Mutagenesis and Protein Engineering


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Презентация на тему: Directed Mutagenesis and Protein Engineering


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№ слайда 1 Directed Mutagenesis and Protein Engineering
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Directed Mutagenesis and Protein Engineering

№ слайда 2 Mutagenesis Mutagenesis -> change in DNA sequence -> Point mutations or la
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Mutagenesis Mutagenesis -> change in DNA sequence -> Point mutations or large modifications Point mutations (directed mutagenesis): Substitution: change of one nucleotide (i.e. A-> C) Insertion: gaining one additional nucleotide Deletion: loss of one nucleotide

№ слайда 3 Consequences of point mutations within a coding sequence (gene) for the protein
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Consequences of point mutations within a coding sequence (gene) for the protein

№ слайда 4 Mutagenesis Comparison of cellular and invitro mutagenesis
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Mutagenesis Comparison of cellular and invitro mutagenesis

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№ слайда 6 General strategy for directed mutagenesis
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General strategy for directed mutagenesis

№ слайда 7 Approaches for directed mutagenesis -> site-directed mutagenesis -> point
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Approaches for directed mutagenesis -> site-directed mutagenesis -> point mutations in particular known area result -> library of wild-type and mutated DNA (site-specific) not really a library -> just 2 species -> random mutagenesis -> point mutations in all areas within DNA of interest result -> library of wild-type and mutated DNA (random) a real library -> many variants -> screening !!! if methods efficient -> mostly mutated DNA

№ слайда 8 Protein Engineering -> Mutagenesis used for modifying proteins Replacements o
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Protein Engineering -> Mutagenesis used for modifying proteins Replacements on protein level -> mutations on DNA level Assumption : Natural sequence can be modified to improve a certain function of protein This implies: Protein is NOT at an optimum for that function Sequence changes without disruption of the structure (otherwise it would not fold) New sequence is not TOO different from the native sequence (otherwise loss in function of protein) consequence -> introduce point mutations

№ слайда 9 Protein Engineering Obtain a protein with improved or new properties
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Protein Engineering Obtain a protein with improved or new properties

№ слайда 10 Rational Protein Design
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Rational Protein Design

№ слайда 11 Site-directed mutagenesis methods
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Site-directed mutagenesis methods

№ слайда 12 Site-directed mutagenesis methods
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Site-directed mutagenesis methods

№ слайда 13 Site-directed mutagenesis methods – Oligonucleotide - directed method
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Site-directed mutagenesis methods – Oligonucleotide - directed method

№ слайда 14 Site-directed mutagenesis methods – PCR based
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Site-directed mutagenesis methods – PCR based

№ слайда 15 Directed Evolution – Random mutagenesis
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Directed Evolution – Random mutagenesis

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№ слайда 17 General Directed Evolution Procedure
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General Directed Evolution Procedure

№ слайда 18 Directed Evolution Library
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Directed Evolution Library

№ слайда 19 Limitation of Directed Evolution
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Limitation of Directed Evolution

№ слайда 20 Typical Directed Evolution Experiment
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Typical Directed Evolution Experiment

№ слайда 21 Evolutionary Methods Non-recombinative methods: -> Oligonucleotide Directed M
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Evolutionary Methods Non-recombinative methods: -> Oligonucleotide Directed Mutagenesis (saturation mutagenesis) -> Chemical Mutagenesis, Bacterial Mutator Strains -> Error-prone PCR Recombinative methods -> Mimic nature’s recombination strategy Used for: Elimination of neutral and deleterious mutations -> DNA shuffling -> Invivo Recombination (Yeast) -> Random priming recombination, Staggered extention precess (StEP) -> ITCHY

№ слайда 22 Evolutionary Methods Type of mutation – Fitness of mutants Type of mutations: Be
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Evolutionary Methods Type of mutation – Fitness of mutants Type of mutations: Beneficial mutations (good) Neutral mutations Deleterious mutations (bad) Beneficial mutations are diluted with neutral and deleterious ones !!! Keep the number of mutations low per cycle -> improve fitness of mutants!!!

№ слайда 23 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
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Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

№ слайда 24 Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)
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Random Mutagenesis (PCR based) with degenerated primers (saturation mutagenesis)

№ слайда 25 Random Mutagenesis (PCR based) Error –prone PCR
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Random Mutagenesis (PCR based) Error –prone PCR

№ слайда 26 Random Mutagenesis (PCR based) DNA Shuffling
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Random Mutagenesis (PCR based) DNA Shuffling

№ слайда 27 Random Mutagenesis (PCR based) Family Shuffling
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Random Mutagenesis (PCR based) Family Shuffling

№ слайда 28 Random Mutagenesis (PCR based)
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Random Mutagenesis (PCR based)

№ слайда 29 Directed Evolution Difference between non-recombinative and recombinative method
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Directed Evolution Difference between non-recombinative and recombinative methods

№ слайда 30 Protein Engineering
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Protein Engineering

№ слайда 31 Protein Engineering
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Protein Engineering

№ слайда 32 Protein Engineering
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Protein Engineering

№ слайда 33 Protein Engineering
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Protein Engineering

№ слайда 34 Protein Engineering - Applications
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Protein Engineering - Applications

№ слайда 35 Protein Engineering - Applications
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Protein Engineering - Applications

№ слайда 36 Protein Engineering - Applications
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Protein Engineering - Applications

№ слайда 37 Protein Engineering - Applications
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Protein Engineering - Applications

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№ слайда 45 Protein Engineering - Applications
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Protein Engineering - Applications

№ слайда 46 Protein Engineering – Applications Directed Evolution
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Protein Engineering – Applications Directed Evolution

№ слайда 47 Protein Engineering – Applications Directed Evolution
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Protein Engineering – Applications Directed Evolution

№ слайда 48 Protein Engineering – Applications Directed Evolution
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Protein Engineering – Applications Directed Evolution

№ слайда 49 Protein Engineering – Applications Directed Evolution
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Protein Engineering – Applications Directed Evolution

№ слайда 50 Protein Engineering – Directed Evolution
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Protein Engineering – Directed Evolution

№ слайда 51 Protein Engineering - Applications
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Protein Engineering - Applications

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