Aim: Preparation of competent cells and their transformation by CaCl2 method.

Calculation of transformation efficiency from the data/plate provided.

In molecular biology, transformation is genetic alteration of a cell resulting from the direct
uptake, incorporation and expression of exogenous genetic material (exogenous DNA) from
its surroundings and taken up through the cell membrane(s).

Figure 1: Method of Chemical Transformation

The first protocol for artificial transformation of E. coli was published by Mandel and Higa in
1970. The procedure showed increased permeability of the bacterial cells to DNA after
treatment with calcium (Ca2+) and brief exposure to an elevated temperature, known as heat
shock. This method became the basis for chemical transformation. In 1983, Douglas
Hanahan published an improved method to prepare competent cells, where optimal
conditions and media for bacterial growth and transformation were identified for higher
transformation efficiency.

 Most species of bacteria, including E. coli, take up only limited amounts of DNA
under normal circumstances.
 In order to transform these species efficiently, the bacteria have to undergo some form
of physical and/or chemical treatment that enhances their ability to take up DNA.
 Cells that have undergone this treatment are said to be competent.
 It has been found that E. coli cells and plasmid DNA interact productively in an
environment of calcium ions and low temperature (0–5°C),and that subsequent heat
shock (37–45°C) is important, but not strictly required. Several other factors,
especially the inclusion of metal ions in addition to calcium stimulate the process.

Mechanism of DNA uptake:

Possibly CaCl2 causes the DNA to precipitate onto the outside of the cells, or perhaps the salt
is responsible for some kind of change in the cell wall that improves DNA binding. In any
case, soaking in CaCl2 affects only DNA binding, and not the actual uptake into the cell.
When DNA is added to treated cells, it remains attached to the cell exterior, and is not at this
stage transported into the cytoplasm. The actual movement of DNA into competent cells is
stimulated by briefly raising the temperature to 42°C.
 A very simple, moderately efficient transformation procedure for use with E. coli
involves resuspending log-phase cells in ice-cold 50 mmol/l calcium chloride at about
10ٰ¹⁰ cells/ml and keeping them on ice for about 30 min.
  Plasmid DNA (0. 1 μg) is then added to a small aliquot (0.2 ml) of these now
competent (i.e. competent for transformation) cells, and the incubation on ice
continued for a further 30 min, followed by a heat shock of 2 min at 42°C.
 The cells are then usually transferred to nutrient medium and incubated for some time
(30 min to 1 h) to allow phenotypic properties conferred by the plasmid to be
expressed, e.g. antibiotic resistance commonly used as a selectable marker for
plasmid- containing cells.
 Finally the cells are plated out on selective medium. The calcium chloride affects the
cell wall and may also be responsible for binding DNA to the cell surface. The actual
uptake of DNA is stimulated by the brief heat shock.
 Typically, efficiencies of 10⁷ to 10⁹ transformants/μg can be achieved depending on
the strain of E. coli
 A large batch of competent cells can be made and store them frozen for future use.
 Unfortunately, some competent cells made rapidly lose their competence on storage.

Preparation of Ultracompetent Cells for Chemical Transformation

Important: All steps in this procedure should be done aseptically in the laminar hood and
under cool conditions.

1. Primary Inoculation: A day before preparing the cells, a single colony of E. coli
DH5α was inoculated from a fresh plate/glycerol stock into 5 mL LB tube and
incubated for 18–20 h at 37°C with vigorous agitation (overnight).
2. Secondary Inoculation: From the above tube, 100 mL LB flask was inoculated with
1% inoculum (i.e., 1 mL in 100 mL) and incubated for 1 and 1⁄2 to 2 and 1⁄2 h at 37°C
with vigorous shaking at around 200 rpm. For efficient transformation, it is essential
that the number of viable cells should not exceed 108 cells/mL. To ensure that the
culture does not grow to a higher density, it is advised that the O.D.600 should be
monitored after regular intervals till it approaches 0.4–0.5.
3. Buffers TFB1,TFB2, centrifuge tubes, eppendorfs were ice chilled.
4.1 mL of overnight culture was inoculated to 100 mL LB flask.
4. O.D.600 was monitored until it reaches 0.2 to 0.6.
5. Culture was transferred to centrifuge tubes and chilled on ice for 10–15 min.
6. Cells were harvested at 4000x g for 10 min at 4°C.
7. Supernatant was decanted.
8. Pellet was resuspended in 1/3 original volume chilled TFB1 buffer.
9. Cells were gently mixed and evenly resuspended till no clumps are visible.
10. Cells were incubated on TFB1 on ice for 15 min.
11. Cells were pelleted by centrifugation at 4000x g for 15 min at 4°C.
12. Supernatant was decanted.
13. Cells were resuspended in 1/25th original volume chilled TFB2 buffer.
14. Cells were incubated on TFB2 buffer on ice for 15 min.
15. Aliquots of 100 µL each were transferred into chilled 1.5 mL eppendorf tubes and
frozen on ice. Stored at – 80°C.

Composition of Buffers used above:

TFB1 Buffer- 30 mM potassium acetate; 10 mM CaCl2; 50 mM MnCl2; 100 mM RbCl2

TFB2 Buffer- 100 mM MOPS; 75 mM CaCl2; 10 mM RbCl; 15% glycerol

Protocol for Heat Shock:
 The transformation mixture was heat shocked by placing the bottom of the tube into a
42°C water bath for 1 min 15 s.
 Tubes were put back on ice for 5 min.
 800 µL of LB (without antibiotic) was added and grown in 37°C shaking incubator
for 1 h.

 Some or all of the transformation was plated onto LB agar containing proper
antibiotic.

 Plates were incubated at 37°C overnight.

 Next day, on the basis of number of colonies obtained, transformation efficiency was
calculated

Figure 2: Procedure followed for transformation

TRANSFORMATION EFFICIENCY:

The transformation efficiency (TE) is defined as the number of transformants generated per
mg of supercoiled plasmid DNA used in the transformation reaction. It is calculated as:
 Numericals to be done:

Ques 1. Calculate the Transformation efficiency from the data provided. Comment on the TE
calculated.

Total volume of the transformation mixture 1 ml

Volume of culture plated 100ml
Volume of plasmid added 5 ml
Concentration of plasmid used 10 mg/ml
Colony forming units on plate 100

Ques 2. Calculate the Transformation efficiency from the data provided. Comment on the TE
calculated.

Total volume of the transformation mixture 1 ml

Volume of culture plated 50ml
Volume of plasmid added 3 ml
Concentration of plasmid used 5 mg/ml
Colony forming units on plate As shown on plate below