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Bernard R. Glick Molecular Biotechnology PowerPoint Presentation
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Bernard R. Glick Molecular Biotechnology

Bernard R. Glick Molecular Biotechnology

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Bernard R. Glick Molecular Biotechnology

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  1. Bernard R. Glick Molecular Biotechnology Text:Molecular BiotechnologyB.R. Glick, J.J. Pasternak and C.L. PattenASM Press, Fourth Edition

  2. Biotech Companies Worldwide in 2004

  3. Fundamentals of recombinant DNA technology

  4. Overview of recombinant DNA cloning procedure

  5. Plasmid pBR322 Strategy for selecting E. coli cells transformed with pBR322

  6. pBR322 Francisco Bolivar Raymond Rodriguez

  7. Screening a gene library with a labeled DNA probe (colony hybridization)

  8. Immunological screening of a gene library (colony immunoassay)

  9. Screening a genomic library for enzyme activity

  10. Tripartite conjugation Electroporation

  11. DNA synthesis and the polymerase chain reaction (PCR)

  12. The chemical synthesis of DNA Gobind Khorana

  13. PCR: first cycle

  14. Second PCR cycle

  15. PCR: thirtieth cycle

  16. 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

  17. Directed Mutagenesis and Protein Engineering

  18. Oligonucleotide directed mutagenesis

  19. Oligonucleotide-directed mutagenesis with plasmid DNA

  20. PCR-amplified oligonucleotide-directed mutagenesis

  21. Error-prone PCR

  22. Random mutagenesis of a cloned target gene

  23. DNA Shuffling

  24. 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

  25. 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.

  26. 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