Bernard R. Glick

Molecular Biotechnology

Text:Molecular Biotechnology

B.R. Glick, J.J. Pasternak and C.L. PattenASM Press, Fourth Edition

Biotech Companies Worldwidein 2004

Fundamentals of recombinant DNA technology

Overview of recombinant DNA cloning procedure

Plasmid pBR322Strategy for selecting E. coli cells transformed with pBR322

pBR322

Francisco Bolivar Raymond Rodriguez

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

Immunological screening of a gene library (colony immunoassay)

Screening a genomic library for enzyme activity

Electroporation Tripartite conjugation

DNA synthesis and the polymerase chain reaction (PCR)

The chemical synthesis of DNA

Gobind Khorana

PCR: first cycle

Second PCR cycle

PCR: thirtieth cycle

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

Directed Mutagenesis and Protein Engineering

Oligonucleotide directed mutagenesis

Oligonucleotide-directed mutagenesis with plasmid DNA

PCR-amplified oligonucleotide-directed mutagenesis

Error-prone PCR

Random mutagenesis of a cloned target gene

DNA Shuffling

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

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.

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