Download E. coli

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