Bacterial transformation is the process by which bacteria take up extracellular DNA from their environment. This DNA can then be incorporated into the recipient bacteria's genome. There are two main methods for artificially inducing bacterial transformation in the laboratory - calcium chloride treatment and electroporation. Calcium chloride treatment makes the bacterial cells competent by creating temporary pores in their cell membranes through which plasmid DNA can enter. The cells are then given a heat shock to help drive the DNA into the cells. Electroporation also creates temporary pores in cells, but uses brief electrical pulses rather than chemicals to do so. Both methods allow exogenous DNA to enter the bacterial cells where it may become incorporated in the genome.

1. Bacterial transformation
Bacterial transformation is the transfer of free DNA released from a donor bacterium into the
extracellular environment that results in assimilation and usually an expression of the newly
acquired trait in a recipient bacterium.
Bacterial transformation is based on the natural ability of bacteria to release DNA which is then
taken up by another competent bacterium.
Competence
The success of transformation depends on the competence of the host cell. Competence is the
ability of a cell to incorporate naked DNA in the process of transformation
Organisms that are naturally transformable spontaneously release their DNA in the late
stationary phase via autolysis.
Bacterial transformation methods
Several bacteria, including Escherichia coli, can be artificially treatedin the laboratory to increase
their transformability by chemicals, such as calcium, or by applying a strong electric field
(electroporation) or by using a heat shock.
 Chilling the cells in the presence of Calcium chloride to make them permeable
 Heat shock treatment at 42°C for 60-120 seconds that causes DNA to enter cells
 Making cells permeable by applying electrical pulses, i.e known as electroporation
Calcium Chloride and heat shock method
Calcium chloride transformation technique is the most efficient technique among the competent
cell preparation protocols. It increases the bacterial cell’s ability to incorporate plasmid DNA,
facilitating genetic transformation. Addition of calcium chlorideto the cell suspension allows the
binding of plasmid DNA to LPS. Thus, both the negatively charged DNA backbone and LPS
come together and when heat shock is provided, plasmid DNA passes into the bacterial cell.
Bacteria can be transformed by using two methods;
 CaCl2 treatment followed by heat shock step
 CaCl2 treatment without using heat shock step

2. DNA being highly negatively charged , cannot pass through bacterial cell membrane.
Bacterial cells can be made competent to take up DNA by making small holes in
bacterial cells by suspending them in a solution containing high concentration of
calcium.
Method
 Prepare a small, overnight culture of the bacteria in LB broth. Grow at 37°C without
shaking.
 About 2 h before you are ready to begin the main procedure, use 1.0 mL of the
overnight culture to inoculate 100 mL of fresh LB broth. This culture is grown with rapid
shaking at 37°C until it reaches roughly 5 x 107 cells/ml. Thus corresponds to an OD650
for our cultures, but you should calibrate this for each of your own strains
 Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic
centrifuge tubes. Cool on ice for 10 mm.
 Re-uspend the cells in 0.2 mL of cold 0.1M CaCl2.
 To each tube add up to 0.1 mg of DNA, made up in a standard DNA storage buffer such
as TE to a volume of 100 mL. Leave on ice for 30 min.
 Transfer the contents of each tube to 2 mL of LB broth in a small flask. Incubate with
shaking at 37°C for 60-90 min.
 Plate 0.1 mL aliquots of undiluted, 10-1 and 10-2 dilutions onto LB plates to which the
antibiotics to be used for selection have been added.
Heat shock is performed at 37–42°C for 25–45 seconds as appropriate for the bacterial strain
and DNA used. For smaller volumes of cells in smaller tubes, the heat-shock interval, which
depends on the surface-to-volume ratio of the cell suspension, should be reduced.
Advantages of Ca+ ion treatment and heat shock:
Ca2+
could develop the interaction between DNA molecules and LPS (lipo-
polysaccharide) of outer membrane. Ca2+
cations improve structural changes in
phosphatidylcholine-lipin bilayers which lead to increased permeability.
Electroporation
Electroporation is a process in which brief electrical pulses create pores in plasma
membrane that allow nucleic acids to enter cell.
Principle:

3. Electroporation allows cellular introduction of large highly charged molecules such as
DNA which would never passively diffuse across the hydrophobic bilayer core.
Electropores were optically imaged in lipid bilayer models like droplet interface bilayers.
During electroporation the lipid molecules are not chemically altered but simply shift
position, opening up a pore which acts as the conductive pathway through the bilayer
as it is filled with water.
Procedure
In laboratory practice, electroporation is done with an appliance that creates an
electromagnetic field in the cell solution known as an electroporator. The cell
suspension is pipetted into a glass or plastic cuvette which has two aluminum electrodes
on its sides.
Applications of electroporation
Electroporation is used in many areas of molecular biology research as well as in the
medical field. Some applications of electroporation include, DNA transfection or
transformation, direct transfer of plasmids between cells, induced cell fusion, trans-
dermal drug delivery, cancer tumor electrochemotherapy and gene therapy.
Advantages and disadvantages
 Versatility: Electroporation is effective with mammalian cell types
 Efficiency: A high percentage of cells are transfected without jeopardizing viability
 Flexibility: A range of cell numbers can be used: from 104 to 106 cells
 Simplicity: Just one kit for all cell types
The major drawback of electroporation is substantial cell death caused by high voltage pulses
and only partially successful membrane repair, requiring the use of greater quantities of cells
compared to chemical transfection methods