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

Other methods of DNA uptake Biolistics Using a DNA coated gold or tungsten fragment to deliver DNA through the cell wall of a plant

Selection of Transformation 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 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