COMPETENT CELL PREPARATION AND TRANSFORMATION

AIM: To prepare competent cells which are capable of taking up foreign plasmid.
PRINCIPLE:
Transformation process allows a bacterium to take up genes from its surrounding environment; that is
transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of
new genetic characteristics.
There are two major parameters involved in efficiently transforming a bacterial organism. The first is the
method used to induce competence for transformation. The second major parameter is the genetic
constitution of the host strain of the organism being transformed. Competent cells are capable of taking
up DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be
competent, it has to maintain a physiological state in which it can take up the donor DNA. Competence
results from alterations in the cell wall that makes it permeable to large DNA molecules.
Transformation & Storage Solution (TSS) enables researchers to prepare competent E. coli in a single
step and to transform the cells without heat-shock. TSS has been reported to be faster and easier than
other methods of producing competent cells, such as the traditional CaCl2 method or other high-
competency protocols.
The CaCl2 method initially produces highly competent E. coli cells, but cell competency decreases
rapidly after storage at -70C for several weeks. Other protocols produce highly competent cells that
have a long storage life, but the procedures are time-consuming, requiring several transformation
buffers or heat-shock steps. In contrast, TSS is a simple, one-step procedure.
Advantages of using TSS
Single-step preparation of competent cells.
Store prepared cells at -70C with little or no loss in transformation efficiency.
Transform cells without heat-shock.
Transformation efficiencies of 106-108/g DNA are typically obtained.
In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in
a waterbath at 42C and further plunging them in ice. This process will take up the DNA into the
bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic.
The presence of an antibiotic marker on the plasmid allows for rapid screening of successful
transformants. Blue white selection (Alpha complementation) can be used to determine which
plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional
gene (lac Z) that encodes for a portion of the enzyme galactosidase. When it transformed into an
appropriate host, one containing the gene for the remaining portion of galactosidase, the intact
enzyme can be produced and these bacteria form blue colonies in the presence of the chromogenic
substrate X gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside).
These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign
DNA into this region results in the loss of the ability to form active galactosidase. Therefore colonies
that carry the plasmid with an insert (Transformants) will remain white and the colonies without the
foreign DNA (Non-Transformants) will remain Blue.
We can also calculate the efficiency of transformation by using the concentration of DNA and number of
transformed colonies.
MATERIAL REQUIRED:
TSS solution (transformation and storage solution)
LB 2.12gms
PEG 10gms
100% DMSO 5ml
1M MgCl2 (pH 6.5) 5ml
Weigh these components, dissolve it completely in 80ml of milliQ water and make up the volume to
100ml with milliQ water. Autoclave and store at 40c (storage period: 1 week)
LB medium:
Weigh 2.5gms in 80 ml Milliq water in 500ml conical flask (head space is necessary for aeration).
Dissolve it completely and make up the volume to 100ml with milliq water. Autoclave and store at room
temperature.
Autoclaved dry 1.5ml eppendrof vials, sterile centrifuge bottles (250ml), dry ice with 100% ethanol.
PROCEDURE:
1. A single colony (DH50 / sbl3) was inoculated into 5ml LB media and grown overnight at 370C with
shaking at 200rpm.
2. 1ml overnight culture was inoculated into 500ml conical flask containing 100ml of sterile LB broth.
3. Culture was incubated at 370c/200rpm till it reaches an OD of 0.3.
4. The flask was removed from the incubator and kept on ice for 20 minutes.
5. The culture was transferred to 250ml sterile centrifuge bottle and centrifuged at 1500rpm for
15minutes at 40C
6. The supernatant was discarded and pellet is restored.
7. Carefully add 10ml of ice cold TSS solution and resuspend slowly.
8. Aliquot 100l of cells in 1.5ml sterile eppendrof vials and immediately flash freeze in either liquid
nitrogen or dry ice containing ethanol(Note: make sure the cells are frozen immediately else the
efficiency might go down).
9. Store at -800C IN cryobox with date of preparation and determine the transformation efficiency
with any plasmid on the same day.
Preparing the competent cells:
Reagent: TSS (transformation and storage solution for chemical transformation)
85% LB medium
10% PEG (wt/vol, MW 8000)
5% DMSO (vol/vol)
50 mM MgCl2 (pH 6.5)
Autoclave or filters sterilize. Store at 40C for <2 weeks.
1. Streak the cell stock on a LB plate (added antibiotic if cells have antibiotic resistant). Incubate the
plate at 370C overnight.
2. Pick a single, well isolated colony and inoculate it into 5ml of LB broth (plus antibiotic). Incubate at
370C overnight with shaking at 220rpm.
3. Transfer 1ml of the standard overnight culture to a sterile 500ml flask containing 100ml of LB
medium (do not add antibiotic at this step). Incubate the cells at 370C with the shaking at 220rpm, until
OD600 reach 0.5; this usually takes 2.0-2.5 hr. heck the OD frequently when it gets beyond 0.2 to avoid
overgrowth.
4. When the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and the collect the
cells by centrifugation at 1500rpm for 5min at 40C.
5. Resuspend the cells in 10ml of ice cold TSS solution. Now the competent cells are ready to be
transformed.
6. Aliquot 150l competent cells to 1.5ml tube. If they are not immediately used, cells can be stored
at 40C for maximum of 6 hr. without significant loss of competency. The same competent cells can also
be stored at -700C for long term storage (pre-treat with liquid nitrogen or dry ice).
7. Competent cells should give a minimum of 1x106 tranformants per g of plasmid DNA.
8. Transformation frequency of frozen cells is 30% of that of the fresh cells, when used within two
months.
Transforming the cells:
1. Add DNA (20 l) ice cold 150l competent cells. Thaw -700C competent cells first by hand
temperature.
(You can transform smaller portions of cells (e.g. 75l or 50l), but you should decrease the volume of
DNA and media proportionately.
2. Incubate on ice for 30 min, with occasional mix.
3. Heat shock at 420C for 2 min.
4. After heat shock, put on ice for 2 min.
5. Add 0.8ml LB broth.
6. Shake and incubate at 370C for 60 min.
7. Plate (<200l) on the appropriate agar plates which contain antibiotic.
8. Incubate plates at 370C, overnight.
OBSERVATION:
The transformed cells were plated on medium containing ampicillin and the negative control plate didnt
show any growth since there were no transformed cells in that plate. The test plate showed growth
indicating the presence of transformed cells.

Positive control showed colonies
Negative control shows no colonies
Test shows few colonies
RESULT:
The organisms were transformed successfully with the plasmid inducing resistance towards ampicillin.
Troubleshooting:
Low efficiency of transformation was observed. It may be due to the improper handling of the cells. The
cells should be thawed and used immediately. Refreezing of the cells can decrease efficiency. Non-
optimized tubes were used. Usage of 17 x 100-mm polypropylene tubes is the best as the heat shock
step is calibrated to the size and material of these tubes. If different tubes are used, the heat transfer to
the cells may not be optimal. An excess DNA was used. No more that 1-10 g of DNA should be used in
100 l of cells. Electroporation must have being a better method.