Aim: Genetic Transformation of E.Coli.

Principle:
In molecular cloning, restriction deficient and usually modification proficient E. coli strains
are commonly used as host. There are number of ways for genetic transformation of E. coli.
However the following protocols are routinely used for transformation: (i) CaCl 2-induced
method of transformation, (ii) High efficiency electrotransformation. Both the protocols are
described precisely in the following sections:
(i) CaCl2-induced method of transformation
The discovery of bacterial transformation was one of the landmark events in biology. It
provided the foundation for modern microbial and molecular genetics. In 1944, Oswald
Avery, Colin Macleod and Maclyn McCarty carried out a critical experiment that
demonstrated that DNA is the active genetic material in bacterial transformation. Bacterial
transformation is a process in which a recipient cell acquires gene from free DNA molecules
in the surrounding medium. Initially, transformation was one of the important mapping
techniques in microbial genetics. Plasmid DNA or bacteriophage DNA may be transferred to
recipient bacteria, which is exceedingly important in modern genetic engineering. At present,
there are many methods available for bacterial transformation. All these are based on the
observations of Mandel and Higa (1970). They showed that bacteria treated with ice-cold
solutions of calcium chloride and then briefly heated could be transfected with bacteriophage
DNA. The same method was subsequently adopted by Cohen et al. (1972) to transform
Escherichia coli with plasmid DNA.
The treatment with CaCl2 induces a transient state of competence in the recipient bacteria
like E. coli cells during which they are able to take up various DNA molecules such as
plasmid DNA, bacteriophage DNA, chromosomal DNA fragments etc. The molecular
mechanisms by which DNA molecule like a plasmid enters competent E. coli cells is not
known clearly. However a great deal of efforts has been made to understand the mechanism
of competence. This appears to result from changes in the cell wall of the bacteria and
probably is associated with the synthesis of cell-wall material at a particular stage of the life
cycle of the bacterium. In the course of developing competence, some particular type of
receptors are either formed or activated on the cell wall; these receptors may be responsible
for binding of free DNA molecules in the medium. The number of active receptors usually
varies from one organism to other. It could be mentioned here that competence usually arises

at a specific stage of growth of a culture, typically late log phage and is highly dependent on
the growth medium and the degree of aeration of culture.
According to the protocol of Mandel and Higa, the efficiency appears to be 105106
transformed colonies/g of supercoiled plasmid DNA in case of E. coli cells. This efficiency
can be increased 100- to 1000-fold by exposing improved strains of E. coli to combinations
of divalent cations for a longer period of time, treating with DMSO, reducing agents and
hexamine cobalt chloride etc.
The transforming plasmids in this experiment are pUC19 (a small, high copy no. E. coli
plasmid, 2,686 bp in length) and pUS19 (a shuttle plasmid vector between E. coli and the
yeast Saccharomyces cerevisiae, 6,110 bp in length, it is a shuttle vector). Because of the
presence of ARS1 and CEN4 modules, pUS19 is also referred to as yeast minichromosome.
Both the plasmids contains ampicillin resistance gene as selection marker. In this
transformation protocol, first we need to prepare competent E. coli cells using CaCl2 solution
followed by transformation using the above plasmids. This is relatively simple and usually
yields 5x106 to 2x107 transformed colonies per microgram of supercoiled plasmid DNA. This
efficiency of transformation is high enough to allow all routine cloning in plasmids.
Requirements
(Media and solutions should be sterile. Sterilization by autoclaving needs to be done at 15 lbs
for 15 minutes)
E. coli DH5 strain, plasmid DNAs: pUC19 and pUS19, LB medium, LA-ampicillin plates
(ampicillin concentration 50-100 g/ml), 100 mM CaCl2, ampicillin stock (25 mg/ml in
water), microfuge tubes and micro tips
Composition of LB medium/litre: Bactotryptone 10 g, Yeast extract 5 g, NaCl 10 g (pH of the
medium should be 7.0-7.2), 15 g agar is required for LA plates.
Procedure

Pick a single colony of E. coli DH5 from a freshly grown plate and transfer it into 20 ml
of LB broth in a 250ml flask. Incubate the culture for 16-20 hrs at 37 0C with vigorous
shaking (200-250 cycles/minute in a rotary shaker).

Aseptically transfer 200l of the above-saturated culture into 20 ml of fresh LB

broth

in a 250ml flask. Incubate the culture with vigorous shaking at 37 oC for 2-3 hrs. To
monitor the growth of the culture, determine the OD590 every one-hour (The OD590 should
be ~ 0.5).

Transfer the above culture to sterile, disposable, ice-cold 50 ml

polypropylene tubes.

Cool the cultures to 0oC by storing the tubes on ice for 10 minutes.

Recover the cells by centrifugation at 5000 rpm for 10 minutes at 4 oC. Decant the media
from the cell pellets. Stand the tubes in an inverted position for 1 min to allow the last
traces of media to drain away. Re-suspend pellet in 10 ml of ice-cold 0.1 M CaCl 2 and
store on ice for 10-15 mins. Recover the cells by centrifugation at 5000 rpm for 10
minutes at 4oC. Decant the fluid from the cell pellets, stand the tube in an inverted
position for 1 min to allow the last traces of fluid to drain away.

Re-suspend the cell pellet in 1 ml of ice-cold 0.1 M CaCl 2. The cells in this stage may be
stored on ice for 12-24 hours. CaCl2 treatment for 2 hours induces considerably a
transient state of competence in the E. coli cells.

Transfer 100 l of the suspension of competent cells to a sterile and pre chilled microcentrifuge tube (1.5 ml capacity). Add plasmid DNA sample (~100 ng in a volume of 5 l
or less) to each tube. (In control experiment, competent bacteria should receive no
plasmid DNA at all.) Mix the content of the tubes gently. Store the tubes on ice for 30
minutes.

With the help of a float, incubate tubes in a circulating water bath that has been preheated
to 42oC. Leave the tubes for exactly 2 minutes without shaking.

Rapidly transfer the tubes to an ice bath and chill the cells for 1-2 minutes.

Add 1 ml of LB broth to each tube. Incubate the cultures for 45-60 minutes at 37 oC to
allow the bacteria to recover and to express the antibiotic resistance marker encoded by
the plasmid.

Take 100 l of transformed cells for plating on each 90 mm LB-antibiotic plates (in this
experiment, LB-ampicillin plates will be used. The conc. of ampicillin being 50 g/ml.
Leave the plates at room temperature until the liquid has been absorbed.

Invert the plates and incubate at 37oC. Transformed colonies should appear in 12-16
hours.