Bernard R. Glick Molecular Biotechnology

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

3. Biotech Companies Worldwide in 2004

6. Fundamentals of recombinant DNA technology

7. Overview of recombinant DNA cloning procedure

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

10. pBR322 Francisco Bolivar Raymond Rodriguez

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

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

13. Screening a genomic library for enzyme activity

14. Tripartite conjugation Electroporation

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

16. The chemical synthesis of DNA Gobind Khorana

17. PCR: first cycle

18. Second PCR cycle

19. PCR: thirtieth cycle

20. 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

21. Directed Mutagenesis and Protein Engineering

22. Oligonucleotide directed mutagenesis

23. Oligonucleotide-directed mutagenesis with plasmid DNA

24. PCR-amplified oligonucleotide-directed mutagenesis

25. Error-prone PCR

26. Random mutagenesis of a cloned target gene

27. DNA Shuffling

28. 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

29. 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.

30. 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