Content uploaded by Farah Deeba
Author content
All content in this area was uploaded by Farah Deeba on Jul 18, 2014
Content may be subject to copyright.
INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
13–353/2014/16–1–132–138
http://www.fspublishers.org
Full Length Article
To cite this paper: Janjua, S., S. Younis, F. Deeba and S.M.S. Naqvi, 2014. High efficiency DNA transformation protocol for Escherichia Coli using
combination of physico-chemical methods. Int. J. Agric. Biol., 16: 132‒138
High Efficiency DNA Transformation Protocol for Escherichia coli using
Combination of Physico-chemical Methods
Safia Janjua, Sidra Younis, Farah Deeba and S.M. Saqlan Naqvi*
Department of Biochemistry, PMAS Arid Agriculture University Rawalpindi, 46300-Pakistan
*For correspondence: saqlan@uaar.edu.pk
Abstract
The present study was designed to achieve high DNA transformation efficiency of E. coli by using the combination of
chemical and physical transformation methods. The effect of low growth temperatures, osmotic agents, reducing agents, field
strength, pre- and post-electroporation heat shock treatments; surfactant and different combinations of some of these factors
were studied in E. coli. Cells grown at low temperature required longer time to reach required density and no improvement in
transformation efficiency observed. Osmotic agents in growth medium changed the growth trend of bacteria and their presence
in transformation mixture proved helpful in achieving up to 108 transformants/µg of plasmid DNA. The addition of β-
mercaptoethanol in growth and transformation mixture improved transformation efficiency by 10 folds compared to control.
Higher field strength of 12.5 kV cm-1 was found to increase the transformation efficiency. Delay in electroporation after
mixing plasmid DNA and competent cells as in case of pre-electroporation heat shock and use of PEG decreased the
transformation efficiency. Pre- and post-electroporation heat shock decreased cell survival as well as transformation
frequency. The use of sucrose, glycine and β-mercaptoethanol (in growth and subsequent transformation media), and field
strength of 12.5 kV cm-1 positively affected the transformation efficiency. © 2014 Friends Science Publishers
Keywords: E. coli; Competent cells; Plasmid; Transformation efficiency
Introduction
Transformation is the genetic alteration of a cell resulting
from the uptake, incorporation and expression of exogenous
DNA that is taken up through the cell envelop. The ability
of bacteria to take up the exogenous DNA in their close
vicinity is referred to as competence (Dreiseikelmann, 1994)
which is genetically programmed physiological state
permitting the efficient DNA uptake. The discovery of
transferring phage DNA (Mandel and Higa, 1970) and
plasmid DNA (Cohen et al., 1972) into E. coli cells set the
stage for molecular cloning, and the quest for the best
artificial mean for transformation of E. coli began. In this
regard scientists studied the effect of various chemicals and
physical treatment to improve the transformation ability of
cells. Chemicals acting as osmotic stabilizers are used in
various studies with the aim to enhance transformation
efficiency using different bacterial species (Thompson et al.,
1998; Arenskotter et al., 2003). Reducing agents can modify
cell surface transport machinery and act as permeabilizer.
Polyethylene glycol (PEG) has been shown to facilitate
uptake of foreign DNA by protoplasts of Gram positive as
well as Gram negative bacterial strains (Hanahan, 1983).
For achieving high transformation efficiency, use of low
growth temperature has been reported that may make the
chemical composition or the physical characteristics of
bacterial membranes more favorable for uptake of DNA
(Inoue et al., 1990).
Electro-transformation is the process of subjecting
living cells to a rapidly changing, high-strength electric field
which results in producing transient pores in their outer
membranes (Szostkova and Horakova, 1998).
Consequently, diffusion and exchange of intracellular and
extracellular components can take place during the lifespan
of the pore. Electroporation is now being widely used to
transfer a variety of macromolecules, including DNA, RNA,
protein, and some chemotherapeutic agents, into cells
(Miller and Nickoloff, 1995). Second most frequently used
physical method for DNA transformation in E. coli is heat
shock treatment. It is an important factor for induction of
free DNA uptake by E. coli cells and inactivates the
restriction enzymes thus suppressing the digestion of
penetrated exogenous DNA by these enzymes (Sambrook
and Russel, 2001a, b).
No doubt some basic methods are used for
bacterial DNA transformation (Sheng et al., 1995;
Neumann et al., 1996) still transformation of ligation
mixture and PCR products is a difficult task in routine
molecular biology lab work. Aim of the present study
was to propose a highly reliable and reproducible
method for the DNA transformation in E. coli by using
combination of chemical and physical methods of
transformation.
High Efficiency E. coli Transformation Protocol / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
133
Materials and Methods
Bacterial Strain, Plasmid and Growth Conditions
E. coli strain DH5α was used in this study. Recombinant
vector pGEMTr (Promega pGEMT® easy vector) with
insert of cp2 viral protein of 4 Kb containing ampicillin
resistance gene was used as transforming DNA. LB (Luria-
Bertani) medium alone and supplemented with 1.5%
sucrose (LB-S), 1% Glycine (LB-G), 5 mM ascorbic acid
(LB-A) and 10 mM β-mercaptoethanol (LB-M) were used
to culture DH5α. Super optimal broth (SOB) with 50 µg/mL
ampicillin was used for selection of transformants. For
making plates, medium was solidified with 1% agar.
Competent Cell Preparation
For competent cells formation, 5 mL overnight starter
culture was inoculated in 100 mL of LB, LB-S, LB-G, LB-
A and LB-M media and allowed to grow at 37ºC and 25ºC
with 250 rpm till growth reached mid log phase (OD600
0.35-0.40). For electroporation, heat shock and PEG studies,
bacterial cells were grown in LB media up to mid log phase.
Competent cells were prepared according to Sambrook and
Russell (2001a). Cell suspension was dispensed into 50 µL
aliquots and stored at -80ºC.
Electroporation and Heat Shock Procedure
Competent cells stored at -80°C were thawed at ice and 1
µL plasmid was directly pipetted over competent cells.
Competent cells were electroporated at 1.1 kV, 1.9 kV and
2.5 kV by BTX, USA model ECM 399 according to
Sambrook and Russell (2001b). These cells were mixed
gently by tapping followed by a heat shock treatment. After
treatment, 950 µL SOC medium was added. The vials were
finally incubated at 37°C for 1 h at 250 rpm in a shaking
incubator. To check the effect of PEG, prior to
electroporation at 2.5 kV transformation mixture was
supplemented with 0%, 5.7%, 11.5% and 17% of PEG
(M.W. 8000) solution to study the combination of
electroporation with PEG. For pre-electroporation heat
treatment, transformation mixture was incubated on ice for 2
min prior to electroporation at 2.5 kV then heat shock was
given at 42oC and 46oC for 2 min. For Post-electroporation
heat treatment, 42 and 46oC heat shock was given to
transformation mixture after electroporation at 2.5 kV.
Reducing and Osmotic Agents
To check the effect of osmotic and reducing agents, 270 mM
sucrose, 1% glycine and 0.03 M β-mercaptoethanol were
used in transformation mixture to observe the change in
transformation yield, before electroporation at 1.9 kV.
Calculation of Transformation Efficiency
The transformation efficiency (transformants/μg DNA) was
calculated as follows:
Statistical Analysis
All the experimental data values were means of log of
transformants/µg plasmid DNA from three independent
experiments and results were presented as mean ± standard
error. The significance of differences between the mean
values was statistically evaluated by one way ANOVA and
2 Factor factorial using the MSTATC. The statistical
significance was all calculated at P < 0.05. Least Significant
Difference (LSD) test was applied where applicable.
Results
Effect of Low Temperature on the Growth of E. coli
Low growth temperature did not improve the transformation
of E. coli DH5α (Table 1). There was no significant
difference of 18oC, 25oC and 37oC on transformation
efficiency of E. coli (Fig. 1).
Effect of Field Strength
In this study, effect of different field strengths was
examined by electroporation at 1.1 kV, 1.8 kV or 2.5 kV
potential differences in a 0.2 cm gapped cell corresponding
to field strength of 5.5 kV cm-1, 9.0 kV cm-1 and 12.5kV.cm-
1 respectively. Pulse duration was kept constant and effect of
field strength was studied (Table 2). Electric shock at 12.5
kV cm-1 gave highest transformation efficiency, 9.0 kV cm-1
medium and least efficiency was obtained with
transformation at 5.5 kV.cm-1 (Fig. 2).
Effect of Osmoticum
The addition of 1.5% sucrose in growth medium and
270 mM sucrose in transformation mixture had
pronounced effect on transformation efficiency (Fig. 3;
Table 3a). Similarly in presence of glycine there was 10-
100 fold increase in transformation efficiency (Fig. 3;
Table 3b).
Effect of Reducing Agents
The addition of 5 mM ascorbic acid in growth medium
acted as growth inhibitor and did not facilitate the
transformation yield as expected (Fig. 4; Table 4a).
However addition of 10 mM β-mercaptoethanol in growth
medium followed by 0.03 mM in transformation medium
resulted in 108 transformants/µg DNA at 25°C (Fig. 4:
Table 4b).
Effect of PEG
Effect of different concentrations (0, 5.7, 11.5 and 17%) of
Janjua et al. / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
134
PEG (M.W. 8000) solution on transformation efficiency
was monitored. Transformation mixture containing 50 µL
competent cells and 1 µL plasmid DNA was made in ice-
cold eppendorf. Then 57% PEG solution and nanopure
water were added in ratio 0 µL:22 µL, 7 µL:15 µL, 15
µL:7 µL, 22 µL:0 µL to transformation mixture for
making 0, 5.7, 11.5 and 17% PEG solution respectively.
After properly mixing, the contents were transferred to
prechilled electroporation cells and shocked at 12.5 kV cm-1.
Results of One-way ANOVA showed that all four
treatments were similar with reference to transformation
efficiency (P>0.05) (Fig. 5; Table 5).
Pre-electroporation Heat Shock Treatment
Results of One-Way ANOVA showed that the three
treatments i.e., electroporation without heat shock,
pre-electroporation heat shock treatment at 42ºC and at 46ºC
were significantly different (P<0.05). LSD results suggested
that the transformation efficiency without heat shock was
greater than either of the pre-electroporation heat shocks at
Table 1: The effect of low temperature on transformation efficiency of E. coli
Treatments
(Temperature)
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
18oC
R1
1.35 × 108
2.00 × 107
14.80
5.10 × 106
25.50
5.10 × 107
R2
2.50 × 107
1.80 × 107
72.00
1.70 × 106
09.40
1.70 × 107
R3
8.30 × 107
2.20 × 107
26.00
8.90 × 105
04.00
8.90 × 106
Average
2.43 × 108
2.00 × 107
37.60
2.56 × 106
12.96
2.56 × 107
25oC
R1
8.62 × 108
9.40 × 107
10.90
5.60 × 106
05.95
5.60 × 107
R2
7.80 × 108
9.10 × 107
11.60
2.40 × 106
03.75
2.40 × 107
R3
8.90 × 107
0.90 × 107
10.10
7.50 × 105
08.33
7.50 × 106
Average
5.77 × 108
6.40 × 107
10.80
2.90 × 106
06.01
2.90 × 107
37oC
R1
1.29 × 108
2.94 × 107
22.79
5.24 × 105
01.78
5.24 × 106
R2
1.56 × 108
4.03 × 107
25.83
4.39 × 105
01.09
4.39 × 106
R3
2.43 × 108
9.56 × 106
03.90
5.16 × 105
05.39
5.16 × 106
Average
1.76 × 108
7.92 × 107
17.50
4.93 × 105
02.75
4.93 × 106
Table 2: Effect of exposure to different electric field strengths for 6 mS on transformation in E. coli DH5α
Field Strength
(kV cm-1)
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
12.5
R1
5.00 × 109
2.46 × 109
49.00
5.70 × 105
2.30 × 10-2
5.70 × 106
R2
6.22 × 108
4.18 × 108
67.00
4.10 × 105
9.00 × 10-2
4.10 × 106
R3
1.10 × 109
8.62 × 108
78.00
4.38 × 106
50.00 × 10-2
4.38 × 107
Average
2.24 × 109
1.25 × 109
64.67
1.79 × 106
20.40 × 10-2
1.79 × 107
9.5
R1
5.00 × 109
2.97 × 109
59.40
4.70 × 105
1.50 × 10-2
4.70 × 106
R2
6.22 × 108
3.80 × 108
61.00
8.70 × 105
22.00 × 10-2
8.70 × 106
R3
1.10 × 109
6.62 × 108
60.00
1.56 × 106
20.00 × 10-2
1.56 × 107
Average
2.24 × 109
1.34 × 109
60.13
9.67 × 105
14.50 × 10-2
9.67 × 106
5.5
R1
5.00 × 109
3.02 × 109
60.40
1.03 × 105
0.34 × 10-2
1.03 × 106
R2
6.22 × 108
6.45 × 108
100.00
1.20 × 105
1.86 × 10-2
1.20 × 106
R3
1.10 × 109
9.96 × 108
90.50
1.30 × 105
1.30 × 10-2
1.30 × 106
Average
2.24 × 109
1.55 × 109
83.60
1.18 × 105
1.17 × 10-2
1.18 × 106
Fig. 1: Effect of different growth temperatures on transformation
efficiency. Low growth temperature did not show any positive
effect on transformation efficiency of E. coli DH5α
Fig. 2: Comparison of transformation efficiency of E. coli DH5α
at different voltages. The highest efficiency was achieved at 12.5
kV cm-1 field strength
High Efficiency E. coli Transformation Protocol / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
135
42ºC or at 46ºC (Fig. 6). Heat shock before electroporation
at either of the temperature decreased the transformation
efficiency by 10 fold than control experiment (Table 6a).
Post-electroporation Heat Shock Treatment
Competent cells and plasmid DNA were shocked at 12.5
kV.cm-1 followed by addition of SOC medium, incubation
for 6 min at 42ºC or 46ºC and then transfer to ice for 5 min
before incubation for growth recovery. The three treatments
were compared with One-way ANOVA and the results
revealed no significant difference among all the three
treatments (P>0.05) (Fig. 6; Table 6b).
Discussion
Results of present study for bacterial growth at low
temperature were in contrast to Inoue et al. (1990); Han et
al. (2003) and Wang et al. (2007), that there was 10-20
folds increase in transformation yield in E. coli when
cells were allowed to grow at 25°C instead of 37°C. In this
study, low growth temperature showed no improvement in
transformation efficiency. Better transformation efficiency
was observed when E. coli DH5α was transformed at high
field strengths. Kinosita and Tsong (1977) reported increase
in the number and radius of pores formed as a function of
increased electric shock intensity, resulting in augmented
transfer of vector DNA to competent cells.
The presence of sucrose in growth medium facilitates
the growth process and early attainment of mid log phase,
therefore exerting positive role in transformation
(Arenskotter et al., 2003; Wang et al., 2007). In the present
study, when electric pulse was applied to transformation
mixture containing 270 mM sucrose, the transient pores
were produced in the cell membranes with the life span of
Table 3a: The effect of osmotic agents (sucrose or glycine) on transformation efficiency of E. coli at 37oC
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
LB
R1
2.20 × 108
7.50 × 107
34.00
6.00 × 105
00.80
6.00 × 106
R2
6.30 × 108
1.05 × 108
16.00
1.00 × 106
00.95
1.00 × 107
R3
4.00 × 108
8.65 × 107
21.00
9.10 × 105
01.05
9.10 × 105
Average
4.16 × 108
8.80 × 107
23.60
8.36 × 105
00.90
8.36 × 106
LB-S
R1
2.30 × 108
8.70 × 107
37.00
8.50 × 105
00.97
8.50 × 106
R2
7.50 × 109
9.60 × 108
12.80
9.50 × 106
00.99
9.50 × 107
R3
7.50 × 108
6.50 × 107
08.66
6.60 × 106
10.10
6.60 × 107
Average
2.80 × 109
3.70 × 108
19.40
5.60 × 106
04.02
5.60 × 107
LB-G
R1
2.90 × 107
1.50 × 107
51.00
2.50 × 106
16.00
2.50 × 107
R2
3.10 × 109
3.70 × 108
28.00
6.10 × 107
07.00
6.10 × 108
R3
8.30 × 108
1.50 × 108
18.00
3.40 × 107
22.60
3.40 × 108
Average
1.31 × 109
3.45 × 108
32.00
3.25 × 107
15.20
3.25 × 108
Table 3b: The effect of osmotic agents (sucrose or glycine) on transformation efficiency of E. coli at 25oC
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
LB
R1
6.80 × 108
1.90 × 108
27.50
1.70 × 106
00.90
1.70 × 107
R2
7.00 × 108
1.56 × 108
22.00
1.00 × 106
00.60
1.00 × 107
R3
7.30 × 108
9.10 × 107
12.40
1.00 × 106
01.00
1.00 × 107
Average
7.03 × 108
1.45 × 108
20.63
1.23 × 106
00.83
1.23 × 107
LB-S
R1
8.10 × 108
1.80 × 108
22.00
4.00 × 107
04.80
4.00 × 108
R2
7.70 × 108
1.70 × 108
22.00
1.10 × 107
06.00
1.10 × 108
R3
7.50 × 108
2.00 × 108
26.70
1.40 × 107
07.00
1.40 × 108
Average
7.76 × 108
1.83 × 108
23.56
2.16 × 107
05.93
2.16 × 108
LB-G
R1
6.30 × 108
1.20 × 108
19.00
1.20 × 107
10.00
1.20 × 108
R2
6.20 × 108
1.10 × 108
18.50
1.40 × 107
12.00
1.40 × 108
R3
5.50 × 108
1.60 × 108
29.00
3.30 × 107
20.60
3.30 × 108
Average
6.00 × 108
1.30 × 108
22.16
1.96 × 107
14.20
1.96 × 108
LB, control cells; LB-S, cells grown in presence of 40 mM sucrose and 270 mM sucrose was added to transformation mixture prior to electric pulse; LB-
G, cells grown in presence of 1 % (w/v) glycine and transformation mixture also contains 1 % (w/v) glycine.
Fig. 3: Effect of sucrose and glycine as growth supplement in LB
growth media and in transformation mixture on E. coli genetic
transformation. LB-S is cells grown with sucrose (1.5%) and 270
mM sucrose in transformation mixture and LB-G is results of
experiment where 1% glycine was used in both growth medium
and in transformation mixture
Janjua et al. / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
136
30 min. The sucrose being higher in concentration in extra
cellular medium than inside the cell cause mass flow of
sucrose through pores from outside to inside of the cell
resulting in concomitant DNA delivery into the cells
(Enyard, 1992).
Glycine acts as inhibitor of bacterial growth but it has
positive impact on transformation yield because its presence
in growth medium interferes in membrane biosynthesis and
replaces L- and D-alanine found in the peptide units of
peptidoglycan rendering membrane more permeable
(Kaderbhai et al., 1997).
Presence of ascorbic acid in growth medium was
supposed to reduce membrane proteins and weaken the
membrane that could have resulted in breaking permeability
barrier for macromolecule, nevertheless our results indicated
that ascorbic acid act only as a growth inhibitor not rather
than a transformation facilitator. The addition of β-
mercaptoethanol (10 mM) in growth medium only, had
neither any effect on transformation efficiency nor on
growth pattern. However, its presence in growth medium
(10 mM) as well as in transformation mixture (0.03 M)
resulted in 10 fold increase in transformation efficiency
(Fig. 4). It has been reported by Puyet et al. (1990) that S.
pneumonia harbor a membrane-bound nuclease required for
transformation. It was therefore, hypothesized that presence
of β-mercaptoethanol in transformation mixture prior to
addition of DNA may prevent inactivation of similar surface
exposed nucleases possessed by E. coli required for
facilitating the process of DNA uptake.
Use of PEG in combination with electroporation is not
Table 4a: The effect of reducing agent, ascorbic acid, on transformation efficiency of E. coli at 37oC
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
LB
R1
4.00 × 108
1.50 × 108
37.00
1.40 × 105
00.09
1.40 × 106
R2
1.30 × 109
3.60 × 108
27.00
1.80 × 105
00.05
1.80 × 106
R3
6.00 × 108
3.00 × 108
50.00
2.00 × 105
00.06
2.00 × 106
Average
7.60 × 108
2.70 × 108
38.00
1.73 × 105
00.20
1.73 × 106
LB-A
R1
2.60 × 108
1.00 × 108
38.00
1.60 × 105
00.16
1.60 × 106
R2
7.60 × 108
1.00 × 108
13.00
1.50 × 104
00.01
1.50 × 105
R3
6.30 × 108
9.90 × 107
25.30
1.70 × 106
01.00
1.70 × 107
Average
1.83 × 108
9.96 × 107
25.40
6.25 × 105
00.40
6.25 × 106
Table 4b: The effect of reducing agent, ascorbic acid, on transformation efficiency of E. coli at 25oC
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
LB
R1
1.40 × 109
3.80 × 108
27.00
9.60 × 106
02.50
9.60 × 107
R2
3.90 × 108
1.20 × 108
30.00
2.80 × 106
02.30
2.80 × 107
R3
6.50 × 108
1.50 × 108
23.00
5.40 × 105
00.36
5.40 × 106
Average
8.13 × 108
2.36 × 108
26.60
4.30 × 106
01.72
4.30 × 107
LB-A
R1
4.10 × 108
5.30 × 107
12.00
2.90 × 105
00.55
2.90 × 106
R2
1.70 × 108
1.60 × 107
09.40
9.40 × 104
06.20
9.40 × 105
R3
5.60 × 108
4.90 × 107
08.70
1.70 × 106
03.40
1.70 × 107
Average
3.80 × 108
3.93 × 107
10.30
6.90 × 105
03.38
6.90 × 106
LB, control cells; LB-A, cells grown in presence of 5 mM L-ascorbic acid in LB medium.
Fig. 4: Effect of ascorbic acid and β-mercaptoethanol
supplemented medium on transformation efficiency in E. coli. LB
represents control cells; LB-A are cells grown with ascorbic acid
(5 mM) and LB-M is indicating results of experiment performed
in presence of β-mercaptoethanol in growth medium as well as in
transformation mixture
Fig. 5: Comparison of E. coli transformation efficiencies with
different PEG cocentrations. The control with 0% PEG gives
better transformation yield compared to average transformation
efficiencies obtained with 5.7, 11.5 and 17%
High Efficiency E. coli Transformation Protocol / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
137
previously reported; hence this combination was also tested,
although with no superior outcome in terms of
transformation efficiency. Tu et al. (2005) and Hanahan et
al. (1991) observed that addition of PEG to the plasmid
DNA and competent cells increased transformation
efficiency but heat shock was not necessary for increasing
number of transformants. PEG functions to condense and
consequently increase the chances of DNA entry to
competent cells (Himeno et al. 1984). PEG facilitates DNA
entry into the chemically prepared competent cells
(Hanahan et al., 1991; Tu et al., 2005) but if other physical
treatment as heat shock was used in combination with PEG,
then efficiency was either same or even declined.
When both heat and electric shocks were administered
in this study, it may have resulted in complete extinction of
membrane potential and decrease in recovery which may be
a consequence of difficulties to the cells owing to exposure
to two different, albeit consecutive shocks. Resultantly the
percentage of cells transformed may have dropped thus
offsetting any increase in ultimate transformation efficiency.
Table 5: Transformation efficiencies in different concentrations of polyethylene glycol
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
0%
R1
1.40 × 109
5.73 × 108
40.90
6.80 × 106
119.00 × 10-2
6.85 × 107
R2
1.90 × 109
4.96 × 108
26.10
3.06 × 106
60.00 × 10-2
3.06 × 107
R3
2.90 × 109
1.95 × 108
6.70
1.20 × 105
06.15 × 10-2
1.20 × 106
Average
2.07 × 109
4.21 × 108
24.57
3.34 × 106
62.00 × 10-2
3.34 × 107
5.7%
R1
1.40 × 109
5.64 × 108
40.20
5.14 × 106
90.00 × 10-2
5.14 × 107
R2
1.90 × 109
4.66 × 108
24.52
4.70 × 105
10.00 × 10-2
4.70 × 106
R3
2.90 × 109
2.25 × 108
7.76
4.00 × 104
01.70 × 10-2
4.00 × 105
Average
2.07 × 109
4.18 × 108
24.16
1.88 × 106
33.90 × 10-2
1.88 × 107
11.5%
R1
1.40 × 109
5.39 × 108
38.50
8.38 × 106
155.00 × 10-2
8.38 × 107
R2
1.90 × 109
4.31 × 108
22.68
6.40 × 105
14.80 × 10-2
6.40 × 106
R3
2.90 × 109
2.49 × 108
8.58
1.40 × 105
00.56 × 10-2
1.40 × 106
Average
2.07 × 109
4.06 × 108
23.25
3.05 × 104
58.00 × 10-2
3.05 × 107
17%
R1
1.40 ×109
3.75 × 108
26.78
4.00 × 104
1.00 × 10-2
4.00 × 105
R2
1.90 × 109
3.81 × 108
20.00
1.00 × 104
0.20 × 10-2
1.00 × 105
R3
2.90 × 109
1.97 × 108
6.79
3.33 × 104
1.69 × 10-2
3.33 × 105
Average
2.07 × 109
3.18 × 108
17.86
2.78 × 104
0.96 × 10-2
2.78 × 105
Table 6a: Transformation efficiency obtained with pre-electroporation heat shocks at different temperatures
Treatments
Replications
No. of cells/mL
No. of cells
survived/mL
% survival
No. of cells
transformed/mL
% Transformants
Transformation
efficiency
Control
R1
1.20 × 1010
4.87 × 109
40.58
2.50 × 105
5.10 × 10-3
2.50 × 106
R2
3.33 × 109
1.24 × 109
37.23
1.53 × 105
12.00 × 10-3
1.53 × 106
R3
1.50 × 109
3.58 × 108
23.86
2.20 × 105
60.00 × 10-3
2.20 × 106
Average
5.61 × 109
2.16 × 109
33.89
2.08 × 105
25.70 × 10-3
2.08 × 106
42ºC
R1
1.20 × 1010
4.01 × 109
33.42
3.00 × 104
0.70 × 10-3
3.00 × 105
R2
3.33 × 109
1.43 × 109
42.90
1.67 × 104
1.20 × 10-3
1.67 × 105
R3
1.50 × 109
7.60 × 107
5.06
1.00 × 104
13.00 × 10-3
1.00 × 105
Average
5.61 × 109
1.84 × 109
27.13
1.89 × 104
5.90 × 10-3
1.89 × 105
46ºC
R1
1.20 × 1010
2.09 × 109
17.41
4.00 × 104
1.90 × 10-3
4.00 × 105
R2
3.33 × 109
7.85 × 108
23.57
2.00 × 104
2.60 × 10-3
2.00 × 105
R3
1.50 × 109
5.90 × 107
3.90
1.00 × 104
13.00 × 10-3
1.00 × 105
Average
5.61 × 109
9.78 × 108
14.96
2.33 × 104
5.80 × 10-3
2.33 × 105
Table 6b: Transformation efficiencies with post-electroporation heat treatment
Treatments
Replications
No. of Cells / mL
No. of Cells
Survived /mL
% Survival
No. of Cells
Transformed /mL
% Transformants
Transformation
Efficiency
Control
R1
4.67 × 109
1.17 × 109
25.05
1.00 × 105
8.50 × 10-3
1.00 × 106
R2
6.00 × 109
1.67 × 109
27.80
1.90 × 105
11.00 × 10-3
1.90 × 106
R3
5.00 × 109
1.43 × 109
28.60
1.50 × 105
10.00 × 10-3
1.50 × 106
Average
5.22 × 109
1.42 × 109
27.15
1.47 × 105
9.80 × 10-3
1.47 × 106
42°C
R1
4.67 × 109
1.39 × 109
29.76
1.60 × 105
11.50 × 10-3
1.60 × 106
R2
6.00 × 109
1.91 × 109
31.80
1.00 × 104
8.30 × 10-3
1.00 × 105
R3
5.00 × 109
1.76 × 109
35.00
1.10 × 105
2.20 × 10-3
1.10 × 106
Average
5.22 × 109
1.69 × 109
32.19
9.33 × 104
7.30 × 10-3
9.33 × 105
46°C
R1
4.67 × 109
1.30 × 109
27.84
6.00 × 104
4.60 × 10-3
6.00 × 105
R2
6.00 × 109
9.40 × 108
15.67
1.00 × 104
1.00 × 10-3
1.00 × 105
R3
5.00 × 109
8.00 × 108
16.00
1.00 × 104
1.25 × 10-3
1.00 × 105
Average
5.22 × 109
1.01 × 109
19.83
2.67 × 104
2.28 × 10-3
2.67 × 105
Janjua et al. / Int. J. Agric. Biol., Vol. 16, No. 1, 2014
138
In order to improve the transformation efficiency, an
effort was made to administer heat shock after
electroporation expecting DNA molecules to move into the
cells through transient pores. The results are shown in Fig.
6, which are in contrast to those of Arenskotter et al. (2003)
in Gordonia polyisoprenivorans, where they observed
enhancement in transformation efficiency with post-
electroporation heat shock treatment at 46ºC for 6 min. This
difference in results might be due to differences in cell wall
composition and structure between G. polyisoprenivorans
and E. coli.
In conclusion, the highest transformation efficiency
was obtained when sucrose was added in growth medium as
well as in transformation mixture. Use of glycine in growth
medium and transformation mixture also give high
transformation efficiency. Thus, osmoticum on the whole
have positive effect on transformation yield. Higher
transformation efficiency was also obtained with β-
mercaptoethanol. Among the three field strengths tested
during this study, 12.5 kV cm-1 proved to be the best field
strength. Use of low temperature, PEG, ascorbic acid and
pre and post electroporation treatments did not show any
positive effect on transformation efficiency.
Acknowledgments
The author acknowledges the financial grant from NRPU-
1909 project funded by Higher Education Commission.
References
Arenskotter, M., D. Baumeister, R. Kalscheuer and A. Steinbuchel, 2003.
Identification and Application of Plasmid Suitable for Transfer of
Foreign DNA to Members of the Genus Gordonia. Appl. Environ.
Microbiol., 69: 4971‒4974
Cohen, S.N., A.C.Y. Chang and L. Hsu, 1972. Nonchromosomal Antibiotic
Resistance in Bacteria: Genetic Transformation of E. coli by R-factor
DNA. Proc. Natl. Acad. Sci. USA, 69: 2110‒2114
Dreiseikelmann, B., 1994.Translocation of DNA across bacterial
membranes. Microbiol. Rev., 58: 293‒316
Enyard, N., S. Sixou, N. Duran and J. Teissie, 1992. Fast kinetics
studies of E. coli electro transformation. Eur. J. Biochem., 209:
431‒436
Han, F., Y. Chu and W. Yu, 2003. Effect of low-temperature culture on the
transformation efficiency of E. coli by electroporation. High Technol.
Lett., 9: 30–34
Hanahan, D., 1983. Studies on Transformation of E. coli with plasmids. J.
Mol. Biol., 166: 557‒580
Hanahan, D., J. Jesse and F.R. Bloom, 1991. Plasmid transformation of E.
coli and other bacteria. Meth. Enzymol., 204: 63‒113
Himeno, M., T. Shimbata, Y. Kawahara, Y. Hanaoka and T. Komano,
1984. Effect of Polyethylene Glycol in Plasmid DNA Solution on
Transformation of CaCl2- Treated E. coli Cells. Agric. Biol. Chem.,
48: 657‒662
Inoue, H., J. Nojima and H. Okayama, 1990. High efficiency transformation
of E. coli with plasmids. Gene, 96: 23‒28
Kaderbhai, N., A. Karim, W. Hankey, G. Jenkins, J. Venning and M.A.
Kaderbhai, 1997. Glycine-induced extracellular secretion of a
recombinant cytochrome expressed in E. coli. Biotechnol. Appl.
Biochem., 25: 53‒61
Kinosita, K. and T.Y. Tsong, 1977.Survival of sucrose-loaded erythrocytes
in the circulation. Biochem. Biophys. Acta., 471: 227‒242
Mandel, M. and A. Higa, 1970. Calcium-dependent bacteriophage DNA
infection. J. Mol. Biol., 53: 159‒162
Miller, E.M. and J.A. Nickoloff, 1995. E. coli electro-transformation. In:
Electroporation Protocols for Microorganisms, pp: 105‒114.
Nickoloff, J.A. (eds.). Humana Press, Totowa, New Jersey
Neumann, E., S. Kakorin, I. Tsoneva, B. Nikolova and T. Tomov, 1996.
Calcium-mediated DNA adsorption to yeast cells and kinetics of cell
transformation by electroporation. J. Biophys., 71: 868‒877
Puyet, A., B. Greenberg and S.A. Lacks, 1990. Genetic and structural
characterization of endA. A membrane-bound nuclease required for
transformation of Streptococcus pneumonia. Mol. Biol., 213:
727‒738
Sambrook, J. and D.W. Russel, 2001a. Agarose gel electrophoresis. In:
Molecular Cloning: a Laboratory Manual, 3rd edition, pp:
1.38‒1.42. Cold Spring Harbor, New York
Sambrook, J. and D.W. Russel, 2001b. Transformation of E. coli by
electroporation. In: Molecular Cloning: a Laboratory Manual, 3rd
edition, pp: 1.105‒1.122. Cold Spring Harbor, New York
Sheng, Y., V. Mancino and B. Birren, 1995.Transformation of Escherichia
coli with large DNA molecules by electroporation. Nucleic Acids
Res., 23: 1990‒1996
Szostkova, M. and D. Horakova, 1998. The effect of plasmid DNA sizes
and other factors on electrotransformation of E. coli JM109.
Bioelectrochem. Bioenerg., 47: 319‒323
Thompson, J.R., E. Register, J. Curette, M. Kurtz and R. Kelly, 1998. An
improved protocol for the preparation of yeast cells for
transformation by electroporation. Yeast, 14: 565‒571
Tu, Z., G. He, K.X. Li, M.J. Chen, J. Chang, L. Chen, Q. Yao, D.P. Liu,
H.J. Shi and X. Wu, 2005. An improved system for competent cell
preparation and high efficiency plasmid transformation using
different E. coli strains. Elect. J. Biotech., 18: 114‒120
Wang, S., Y. Luo, X. Yi, W. Yu, Z. Xu, X. Ma, J. He and Q. Liu, 2007. A
Highly Efficient and Highly Reliable Protocol for Transformation of
E. coli by Electroporation. J. Rapid Meth. Automation Microbiol. 15:
253‒258
(Received 18 March 2013; Accepted 12 August 2013)
Fig. 6: Effect of pre- and post-electroporation heat shock on
transformation efficiency. Control (transformation with
electroporation) gave better efficiency than electroporation
combined with heat shock at any of above mentioned temperatures
