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Transformation of Bacteria • Transformation occurs when bacteria incorporate foreign DNA into their cell from the environment. Since bacteria is surrounded by a phospholipid bilayer the cell must become competent to receive DNA • Electroporation is a process of inducing the cell to uptake DNA • CaCl2 (calcium chloride) is commonly used as a transforming agent in order to make the cell “chemically competent”. Transformation Procedure • E. coli is harvested in a logarithmic growth phase from an agar plate • E. coli are then exposed to CaCl2 and stressed in ice water • Increases competency • Plasmids are added to the mix • Containing Ampr or bla & a marker such as GFP • Mix is incubated on ice for 15min then transferred to 42oC bath for 2 min to heat shock the bacteria • Increases efficiency of uptake • LB broth is added to suspended colonies and allowed to equilibrate for 10 min • New recombinant cells are plated on fresh plates and incubated at 37oC overnight • On Amp+ and Amp- plates Other methods of DNA uptake • Eukaryotic cells • Transfection using lipids • Plasmids sealed in tiny lipid vesicles are fused with the plasma cell membrane where they release DNA into the cell • Shuttle plasmids are plasmids engineered to infect eukaryotic cells. • A selectable marker (antibiotic resistance gene) such as neomycin and a promotor from a mammalian virus to aid in DNA insertion • CMV (cytomegalovirus) is a human virus that effectively infects many different types of cells • Biolistics • Using a DNA coated gold or tungsten fragment to deliver DNA through the cell wall of a plant Other methods of DNA uptake • Color • GFP or GRP are fluorescent markers • pUC18 contains the lacZ gene that grows blue colonies when grown on galactose medium • An MCS site is incorporated into the lac Z gene to knock out the lacZ gene when DNA is successfully inserted making the colonies white • Antibiotic resistance • Added into cloning vectors • Lethal genes • pJET1.2 includes a gene for a lethal protein the kills E. coli • An MCS site is inserted within the gene so that it is turned off when DNA is successfully inserted Selection of Transformation • Transformation efficiency is measured colony forming units per micrograms of plasmid DNA (CFU/mg) • Efficiency is affected by all variables • Bacteria used, Electroporation method, Heat shock temp and timing, Plasmid amount, size, and type, … • Calculating efficiency… 10ng is transformed into 500ml. 10ml of that solution is then spread onto an agar plate and incubated and transformed cells counted. • Calculation • DNA spread (mg) = (volume spread(ml) x DNA wt(mg) total transformation volume (ml) • 10(ml) x 0.05(mg) = 0.001(mg) 500(ml) • CFU/mg is # of colonies/DNA spread • 196/0.001(mg) = 1.96 x 105 CFU/mg Transformation efficiency