B. Tech. (Biotechnology) III Year V th Semester Unit II Lecture 2 B. Tech. (Biotechnology) III Year V th Semester EBT-501, Genetic Engineering

EBT 501, Genetic Engineering Unit I Gene cloning -concept and basic steps; application of bacteria and viruses in genetic engineering; Molecular biology of E. coli and bacteriophages in the context of their use in genetic engineering, Cloning vectors: Plasmid cloning vector PBR322, Vectors for cloning large piece of DNA; –Bacteriophage-l and other phage vectors; Cosmids, Phagemids; YAC and BAC vectors, Model vectors for eukaryotes – Viruses, Unit II Restriction modification, enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene cloning, PCR technology for gene/DNA detection, cDNA, Use of Agrobacterium for genetic engineering in plants; Gene libraries; Use of marker genes. Cloning of foreign genes: DNA delivery methods -physical methods and biological methods, Genetic transformation of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation, Unit III Gene library: Construction cDNA library and genomic library, Screening of gene libraries – screening by DNA hybridization, immunological assay and protein activity, Marker genes: Selectable markers and Screenable markers, nonantibiotic markers, Gene expression in prokaryotes: Tissue specific promoter, wound inducible promoters, Strong and regulatable promoters; increasing protein production; Fusion proteins; Translation expression vectors; DNA integration into bacterial genome; Increasing secretions; Metabolic load, Recombinant protein production in yeast: Saccharomyces cerevisiae expression systems; Mammalian cell expression vectors: Selectable markers; Unit IV Origins of organismal cloning in developmental biology research on frogs; nuclear transfer procedures and the cloning of sheep (Dolly) & other mammals; applications in conservation; therapeutic vs. reproductive cloning; ethical issues and the prospects for human cloning; Two-vector expression system; two-gene expression vector, Directed mutagenesis; transposon mutagenesis, Gene targeting, Site specific recombination Unit V General principles of cell signaling, Extracellular signal molecule and their receptors, Operation of signaling molecules over various distances, Sharing of signal information, Cellular response to specific combinations of extracellular signal molecules; Different response by different cells to same extracellular signal molecule, NO signaling by binding to an enzyme inside target cell, Nuclear receptor; Ion channel linked, G-protein- linked and enzyme-linked receptors, Relay of signal by activated cell surface receptors via intracellular signaling proteins, Intracellular signaling proteins as molecular switches, Interaction between modular binding domain and signaling proteins, Remembering the effect of some signal by cells.

DNA Modifying Enzymes Restriction Enzymes The restriction/modification system functions as a type of immune system for individual bacterial strains, protecting them from infection by foreign DNA (e.g. viruses).

W. Arber and S. Linn (1969) Plating efficiencies of bacteriophage lambda (l phage) grown on E. coli strains C, K-12 and B: K-12 and B were protected from bacteriophage. The DNA of phage which had been grown on strains K-12 and B were found to have chemically modified bases which were methylated. Additional studies with other strains indicate that different strains had specific methylated bases. Typical sites of methylation include the N6 position of adenine, the N4 position of cytosine, or the C5 position of cytosine. Methylation occurres at very specific sites in the DNA

Enzymes used in recombinant DNA technology Nucleases Restriction endonucleases S1 nucleases Lamda exonucleases E.coli exonucleases III Exonuclease Bal31 Ligases Polymerases Reverse transcriptase E.coli DNA Polymerase I (klenow fragment) DNA modifying enzymes Alkaline Phosphatase T4 Polynucleotide kinase Terminal deoxynucleotidyl transferase

Nucleases Ligases Restriction endonucleases S1 nucleases Type II cut DNA at specific sites S1 nucleases Used for removal of single stranded protrusions from 5 prime and 3 prime ends Lamda exonucleases Removes one nucleotide from 5 prime end E.coli exonucleases III Removal of single nucleotide at a time from 3 prime end Exonuclease Bal31 to make blunt ends shorter from 5 prime and 3 prime ends Ligases To seal the nicks by forming phosphodiester bond

Polymerases DNA modifying enzymes Reverse transcriptase To make cDNA from mRNAs E.coli DNA Polymerase I (klenow fragment) To make protruding end double stranded by extending short strands DNA modifying enzymes Alkaline Phosphatase To remove 5 prime phosphate from DNA T4 Polynucleotide kinase For adding phosphate group to 5 prime free OH end Terminal deoxynucleotidyl transferase For addition of ss sequence to 3 prime end of blunt ended fragments