Transcript Transformation

Transformation
 Transformation :is a process in which cells take up foreign DNA from their
environment.
bacteria take up exogenous (foreign) DNA and produce
the genetic products (proteins) encoded in the foreign
DNA.
Under proper conditions, a cell that is incubated with
plasmid DNA can absorb the plasmid into its
cytoplasm.
E.coli HB 101 strain is an excellent host for cloning experiment.
because they do not have any natural antibiotic resistance .
Transformation enables inexpensive and reliable production of
important medical products such as insulin, human growth
hormone, and other replacement hormone and gene therapies.
For transformation to occur, bacterial cells must be in a particular
physiological state, referred to as competency.
Much current research in molecular biology involves the
transformation of E. coli, an organism that does not naturally enter
a state of competency.
E. coli can artificially be made competent when treated with
chloride salts of the metal cations calcium, magnesium .
DNA and bacteria are kept on ice for half an hour to enable the
DNA to increase interactions between calcium ions and the
negative components of the cell.
Characteristics of plasmid to be suitable for the
transformation process:In genetic research, plasmids must be large enough to
manipulate, small enough for the cell to take up, and include a
marker gene after the targeted gene.
The marker gene allows the researcher to readily identify
which cells successfully acquired the new genetic material.
For example, if a phosphorescence gene follows the gene for
insulin production, then glowing cells successfully took up the
plasmid and can produce insulin
 Note: The process of Genetic engineering is made effective by the
action of two enzymes namely, Restriction enzymes and
ligases.
 Restriction enzymes, called molecular scissors are precisely
cut the desired DNA and its carrying vector, at
specific sites ,
 whereas the ligase enzyme, called Molecular glue are
pasted these DNA fragments into the carrying
STEPS OF BACTERIAL TRANSFORMATION
1. COMPETENCY
2. TRANSFORMATION
3. RECOVERY PERIOD
4. GROWTH & ISOLATION
After make cell competent we make heat shock
Then the tube in which the cells are kept is put at 42°C for
90 seconds (this time can be adjusted depending on the
size of the tube) .
This action is called the 'Heat shock.
Immediate chilling on ice ensures closure of pores.
3- RECOVERY PERIOD
• Allow the cell to regain strength and start to
multiply Necessary to allow expression of the
antibiotic
• resistance gene on the plasmid and insert.
• Add sterile luria broth.
• Move tubes to water bath at 37oc for 30min.
4- Growth & Isolation`

Mark four Luria Broth agar plates
• This concept can be demonstrated by plating the
same volume of recovered cells on selective and
nonselective agar medium.
• The nonselective medium will have many more
growing cells since all the untransformed cells
survive, in addition to the transformed cells.
The nonselective bacterial agar plates will be covered
heavily with untransformed cells, forming a “lawn”,
in contrast to individual colonies obtained on the selective
agar plate.
Transformation Efficiency: The number of cells transformed per 1 microgram
(μg) of DNA is known as the transformation
efficiency.
 In practice, small amounts of DNA are used (5 to
100 nanograms, ng) since excessive DNA (>100 ng)
inhibits the transformation process
Too little DNA can result in low transformation
efficiencies
Calculate the transformation efficiency:-
Transformation Efficiency = no. of colonies
observed/ µg Plasmid
NOTE THAT EACH COLONY ORIGINALLY GREW FROM ONE TRANSFORMED
CELL
IN THIS LAB WE WILL PERFORM
TRANSFORMATION OF E COLI
• PUC8 = AMPICILLIN RESISTANT PLASMID WILL BE INSERTED INTO E COLI
4 plates
 Luria Broth  Luria Broth +
 AMP Broth  AMP Broth +
No antibiotic, no plasmid
No antibiotic, With plasmid
antibiotic, No plasmid
antibiotic, With plasmid
WHAT DO YOU EXPECT
• LURIA BROTH -
NO ANTIBIOTIC, NO PLASMID
NORMAL GROWTH = LAWN OF BACTERIA
• LURIA BROTH +
NO ANTIBIOTIC, WITH PLASMID
NORMAL GROWTH = LAWN OF BACTERIA
• AMP BROTH -
ANTIBIOTIC, NO PLASMID
NO GROWTH, ALL KILLED
• AMP BROTH +
ANTIBIOTIC, WITH PLASMID
INDIVIDUAL COLONY GROWTH OF TRANSFORMED RESISTANT
BACTERIA
RESULTS