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Bridge Class Routing vectors to Genetic Engineering Introduction to Vectors Defining the purview of genetic engineering? 1. 2. 3. 4. 5. 6. 7. Identification of genomic sequences • Reverse/Forward genetics Isolation of genomic sequences • PCR, RT-PCR, genomic/cDNA libraries Verification of genomic sequences • Sequencing technologies Manipulation of genes and genomic sequences • Cutting, pasting, changing (Site directed mutagenesis) • Tools: Enzymes Assorting combinations from unrelated species • Tools: Enzymes Cloning of rDNA • Making use of vectors and strains Expressing heterologous biomaterial (proteins of therapeutic or economic value in microorganism to provide proof of concept, pilot scale, industrial level Considerations prior to cloning (Plasmid Vectors) PCR amplified Forced ‘A’ tailing Pfu polymerase Source of the genomic fragment Released thru Restriction (frm another vector) Taq polymerase • Linearaisation of vector with compatible RE •CIAP Treatment of vector Choice w.r.t Ligation strategies TA Cloning TOPO-TA Cloning S1 nuclease Blunt end Cloning •Linkers and adaptors •Homopolymeric tailing of vector and insert Cohesive end Cloning Standard methodology of cloning Pipeline downstream of Ligation Transformation •Heat shock method •Electro-transformation Preparation of Host: Competent cells (Chemi compis or electro-compis) •CaCl2 method (Mandal and Higa, 1970) •RbCl2 Method (Hanahan, 1983) •Other protocols (eg For electro-competent, glycerol) Ligation mix (mixture of recombinant and nonrecombinant plasmids) A competent E. coli strain E.coli Transformation: Introduction of DNA into host cells Classical definition: Natural uptake of naked ds DNA by bacterial cells. •Fred Griffiths (1928) Streptococcus pneumoniae (a.k.a. Pneumonococcus or Diplococcus) •Avery, McCarty and MacLeod (1944) proved that DNA is the transforming principle . Competence •Only competent cells can take up DNA from external mileu •Competent state induced in response to nutritional shut down •Not all bacteria are competent, all the time (E. coli not naturally competent) Natural competence Artificially induced E. coli K12: laboratory host for rDNA Ensuring Safety in Molecular Biology laboratory/human health/ environment Outcome of Asilomar Conference (Center on California’s Monterey Peninsula 1976) Scientific fraternity realizing the importance of working with “safe” strains of bacteria, viruses and vectors Containment to be made a part of expt design, level of containment proportional to risk involved/biohazard (Risk assessment and analysis) NIH formed the Recombinant DNA Advisory Committee (RAC) It took 1 year (1976) before the first “safe” (EK2 category) line of E. coli was released That year, RAC released a set of guidelines requiring the use of safe bacteria