Bernard R. Glick Molecular Biotechnology Text: Molecular Biotechnology B.R. Glick, J.J. Pasternak and C.L. Patten ASM Press, Fourth Edition

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Text of Bernard R. Glick Molecular Biotechnology Text: Molecular Biotechnology B.R. Glick, J.J. Pasternak...

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  • Bernard R. Glick Molecular Biotechnology Text: Molecular Biotechnology B.R. Glick, J.J. Pasternak and C.L. Patten ASM Press, Fourth Edition
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  • Biotech Companies Worldwide in 2004
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  • Fundamentals of recombinant DNA technology
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  • Overview of recombinant DNA cloning procedure
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  • Plasmid pBR322 Strategy for selecting E. coli cells transformed with pBR322
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  • pBR322 Francisco Bolivar Raymond Rodriguez
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  • Screening a gene library with a labeled DNA probe (colony hybridization)
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  • Immunological screening of a gene library (colony immunoassay)
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  • Screening a genomic library for enzyme activity
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  • Electroporation Tripartite conjugation
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  • DNA synthesis and the polymerase chain reaction (PCR)
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  • The chemical synthesis of DNA Gobind Khorana
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  • PCR: first cycle
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  • Second PCR cycle
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  • PCR: thirtieth cycle
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  • PCR amplification of full length cDNA. The terminal transferase activity of reverse transcriptase adds mostly dCs to the ends of each full length first strand cDNA
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  • Directed Mutagenesis and Protein Engineering
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  • Oligonucleotide directed mutagenesis
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  • Oligonucleotide-directed mutagenesis with plasmid DNA
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  • PCR-amplified oligonucleotide-directed mutagenesis
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  • Error-prone PCR
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  • Random mutagenesis of a cloned target gene
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  • DNA Shuffling
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  • Addition of a disulfide bond between the N and C termini of B. circulans xylanase stabilizes the protein. The activity at room temperature is doubled and it is largely protected against inactivation at high temperature
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  • Engineering a calcium-independent subtilisn. Native enzyme loses its activity when the calcium-binding loop is deleted. After random mutagenesis, several mutants with low activity are isolated. These are combined into one mutant construct with high activity.
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  • Altering multiple enzyme properties at once. Start with multiple copies of a Bacillus subtilisn gene. Error-prone PCR and then DNA shuffling. Test for high activity at room temp and then stability at high temp