Folia Microbiol
DOI 10.1007/s12223-013-0267-1
Improved method for high-efficiency electrotransformation
of Escherichia coli with the large BAC plasmids
Jana Nováková & Anita Izsáková & Tomáš Grivalský &
Christian Ottmann & Marian Farkašovský
Received: 16 January 2013 / Accepted: 28 June 2013
# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013
Abstract High transformation competency of Escherichia
coli is one of the critical factors in the bacterial artificial
chromosome (BAC)-based DNA library construction. Many
electroporation protocols have been published until now, but
the majority of them was optimized for transformation of
small plasmids. Large plasmids with a size above 50 kbp
display reduced transformation efficiency and thereby require
specific conditions in the preparation and electroporation of
electrocompetent cells. In the present work, we have opti-
mized the parameters critical to the application of BAC
DNA electrotransformation into E. coli. Systematic evaluation
of electroporation variables has revealed several key factors
like temperature of growth, media supplements, washing
buffer, and cell concentration. Improvements made in the
transformation protocol have led to electrocompetent cells
with transformation efficiency up to 7×108 transformants
per microgram of 120 kbp BAC plasmid DNA. We have
successfully used in-house prepared competent cells, the qual-
ity of which is comparable with those produced by different
companies, in the construction of metagenomic libraries from
the soil. Our protocol can also be beneficial for other applica-
tion with limited DNA source.
Introduction
Methods for transfer of exogenous DNA into cells play a
crucial role in genetics and molecular biology. A number of
J. Nováková : A. Izsáková : T. Grivalský : M. Farkašovský (*)
Department of Microbiology, Institute of Molecular Biology,
Slovak Academy of Sciences, Dúbravská cesta 21,
84551 Bratislava, Slovakia
e-mail: marian.farkasovsky@savba.sk
C. Ottmann
Chemical Genomics Centre of the Max-Planck-Society,
Otto-Hahn-Str. 15, 44227 Dortmund, Germany
methods have been developed to artificially transfer DNA into
cells. Two major techniques, chemotransformation and elec-
troporation, have been used to introduce DNA into bacterial
cells. Several other methods were also described, including
transformation of protoplasts and spheroplasts, the freeze and
thaw technique, biolistic transformation, sonoporation,
liposome-mediated DNA transfer, chitosan-mediated transfor-
mation, and tribos transformation. However, most of them
have only a narrow range of use, limited transformation effi-
ciency, or require expensive equipment (Aune and Aachmann
2010). Historically oldest method, chemical transformation,
uses treatment with polyvalent cations and heat shock for
transient opening gated membrane channels. Since the first
demonstration of chemical transformation method (Mandel
and Higa 1970), many different procedures have been devel-
oped with variable transformation efficiencies. A detailed
analysis of factors critical to chemical transformation is pro-
vided in the article by Hanahan et al. (1991). Preparation of
chemically competent cells is relatively simple, no special
equipment is required, and the transformation efficiency can
reach up to 109 transformants per microgram of plasmid
DNA.
Wong and Neumann (1982) first described that electric
impulses in the intensity range of 5–10 kV/cm notably in-
creases the input of linear and circular DNA into mouse
lyoma cells and formation of stable transformants. This
technique has been termed electroporation and was demon-
strated for the first time in prokaryotic organism 1 year later
(Shivarova et al. 1983). The authors have observed that the
high electric field increased the PEG-mediated transforma-
tion of Bacillus cereus protoplasts. The transformation of
Escherichia coli with plasmid DNA by electroporation was
reported for the first time by Fiedler and Wirth (1988). Under
conditions described in this paper, E. coli was transformed at
a rate of 1×105 per microgram of plasmid pBR322.
The detailed mechanism behind electrotranslocation of
DNA through the membrane is not yet known. Several

laboratories have proposed two-step process of electropora-
tion (Xie et al. 1990; Sukharev et al. 1992). At first, the
electric field forms primary pores, whereas in the following
step, DNA is driven by electrophoretic force through the
pore, the size of which is modified by the interaction with
DNA. Recent methodological developments in membrane
research revealed that the electropermeabilization of the
membrane is a more complex process (Teissie et al. 2005).
To make electroporation feasible for genomic (or cDNA)
library production, improvement was required in transfor-
mation efficiency. Optimized protocol for the electroporation
of E. coli (Dower et al. 1988) has yielded transformation
rates in the range of 109 to 1010 transformants per microgram
of small plasmid DNA like pBR322 and pUC18. Efficiency
of electroporation is influenced by factors such as composi-
tion of growth media, temperature, phase of growth, washing
buffer, electric field strength, and time constant of electric
pulse. Significant reduction in transformation efficiency with
increasing size of DNA was previously described (Leonardo
and Sedivy 1990; Woo et al. 1994; Sheng et al. 1995;
Szostková and Horáková 1998). In these reports, it was
observed that transformation rates dropped by up to two
orders of magnitude depending on the size of the used
plasmid. The efficiency of electroporation with large DNA
is also dependent to a great extent on the used E. coli strain
(Sheng et al. 1995).
Our research activity is focused on the construction of
metagenomic libraries containing large environmental DNA
inserted in an F factor-based BAC vector and their screening
for novel secondary metabolites. The tremendous diversity
of uncultured microorganisms in soil and other environments
provides a rich repository of these natural products. Genes
encoding enzymes required for biosynthesis of secondary
metabolites are clustered on coherent piece of DNA, which
range in size from approximately 20 to 150 kbp. Despite the
progress in sequencing methodologies (next-generation se-
quencing), discovery of such long sequences by direct se-
quencing is still not possible (Mende et al. 2012). Therefore,
the construction of BAC libraries from environmental DNA
is still up to date for some application. With respect to the
limited amount of pure environmental DNA and low effi-
ciency of large fragments ligation, the high transformation
rate is a crucial factor for construction of the representative
metagenomic library. Electrocompetent cells yielding over
1×1010 transformants per microgram of plasmid DNA
(pUC18) are commercially available from many vendors,
but the protocol about their preparation is not accessible.
Published protocols which we tried did not reach this high
transformation rate. In light of high consumption of compe-
tent cells during library construction and the cost of such
ultra-high-competency cells, we decided to prepare adequate
substitution in-house. To fulfill this aim, we optimized in our
present investigation the different steps in the preparation of
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electrocompetent cells and electroporation condition with
emphasis on transformation of large-size BAC plasmids.
Material and methods
Bacterial strains, plasmids, and culture media
E. coli DH5α [F- endA1 glnV44 thi-1 recA1 relA1 gyrA96
deoR nupG Φ80lacZΔM15 Δ(lacZYA-argF)U169 hsdR17(rK-
mK+) λ– ] cells (Meselson and Yuan 1968) were cultivated in
various growth media: LB (1 % bacto trypton, 0.5 % yeast
extract, 0.5 % NaCl), TB (1.2 % bacto trypton, 2.4 % yeast
extract, 72 mmol/L K2HPO4, 17 mmol/L KH2PO4, 0.4 %
glycerol), 2xYT (1.6 % bacto trypton, 1 % yeast extract,
0.5 % NaCl), SOB- (2 % bacto trypton, 0.5 % yeast extract,
10 mmol/L NaCl, 2.5 mmol/L KCl), SOB (SOB-+10 mmol/L
MgSO4 and 10 mmol/L MgCl2), SOC (SOB+20 mmol/L
glucose), and SOC- (SOB-+20 mmol/L glucose). Media were
purchased from BD Biosciences (BactoTM, Franklin Lakes,
NJ, USA) and chemicals from Sigma-Aldrich (BioUltra, St.
Louis, MO, USA). Concentration of kanamycin used in selec-
tive LB agar media was 30 μg/mL. E. coli DH10B [F- endA1
recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15
araD139 Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC) λ-]
(Casadaban and Cohen 1980), BL21(DE3) [F- ompT gal dcm
lon hsdSB(rB- mB-) λ(DE3)] (Studier and Moffatt 1986), and
BW25141 [F-, Δ(araD-araB)567, ΔlacZ4787(::rrnB-3),
Δ(phoB-phoR)580, λ-, galU95, ΔuidA3::pir+, recA1,
endA9(del-ins)::FRT, rph-1, Δ(rhaD-rhaB)568, hsdR514]
(Datsenko and Wanner 2000) were used for comparison. The
plasmids used in this study were pBluescript II KS (Alting-
Mees and Short 1989) and a random clone of a metagenomic
library bearing 120 kbp insert in BAC plasmid (pFML1,
unpublished results).
Preparation of competent cells
Protocol from Dower et al. (1988) was employed as a point
of departure to explore each step of competent cells prepa-
ration for potential improvement. Optimal condition of par-
ticular step was thereafter used in the next step tuning.
Overnight culture grown in LB medium at 37 °C was diluted
in growth medium and incubated at 37 °C or 21 °C with
shaking at 220 rpm to an A600 between 0.2 and 0.9. Cells
were then chilled on ice for 20 min and centrifuged for
20 min at 800 g and 4 °C. Pellets were washed two times
with ice-cold washing solution and centrifuged at the above
conditions. The cells were resuspended in the washing solu-
tion remaining after decantation in the centrifugation tube,
and the volume was then adjusted to give the final concen-
tration of the cells between 1.25 to 7.5×1010 per milliliter
(A600 =25–150). This cell suspension was either used
Folia Microbiol
immediately for electroporation or frozen in liquid nitrogen
and stored at −70 °C.
Electroporation
Electroporation was carried out using an ECM 630 exponen-
tial decay wave electroporation system (BTX Harvard appa-
ratus, Holliston, MA, USA). Electroporation cuvettes
(0.2 cm, Bio-Rad, USA) were chilled on ice, and 50 μL
competent cells was mixed on ice with plasmid DNA. The
suspension was placed into electroporation cuvette and
electroporated at 7.7–12.5 kV/cm, 25 μF and 100–700Ω,
respectively. Nine hundred fifty microliters of ice-cold LB
(or SOC) medium was added immediately after the pulse
application. The suspension was transferred into the tube and
cultivated at 37 °C for an 1 h. Each experiment was repeated
at least three times, and appropriate dilutions were spread on
LB agar media containing 30 μg/mL kanamycin. The plates
were grown at 37 °C overnight. Survival rate was determined
by plating dilutions of cells on nonselective media (LB)
before and after the delivery of electric pulse. Our final
protocol for competent cells preparation and electroporation
is summarized in Table 1.
Results and discussion
A bias in the transformation against large DNA molecules
constitutes a serious obstacle to the construction of represen-
tative BAC libraries containing a heterogeneous population
of environmental DNA inserts. We therefore analyzed con-
ditions of competent cells preparation and electroporation
parameters to improve the transformation rate of E. coli with
large DNA. We have chosen E. coli DH5α, widely used for
the construction of diverse DNA libraries, as a reference
strain in optimization experiments.
Effect of culture condition on transformation efficiency
Media composition has direct influence on growth rate and
cell content. We have tested the impact of different media on
the transformation efficiency of E.coli DH5α. The cells
(100 mL medium) were grown at 37 °C until they reached
A600 =0.5, washed twice with 15 mL 10 % glycerol and
concentrated to a final concentration of 5×1010 per milliliter.
All centrifugation steps were performed at 4 °C. One hun-
dred nanograms of 120 kbp pFML1 plasmid, random
metagenomic library clone, was mixed with 50 μL of cell
suspension, transferred to 0.2 cm cuvette and electroporated
at 12.5 kV/cm, 200-Ω parallel resistor and 25-μF capaci-
tance. The relative high concentration of DNA was selected
to mimic conditions, which we use in the metagenomic
library construction to limit individual transformations to a
Table 1 Protocol for the preparation and electroporation of
electrocompetent E. coli cells
Steps
Preparation of 1. Grow cells at 21 °C with vigorous shaking
electrocompetent in 100 mL SOC medium with 0.2 mol/L
cells sucrose until they reach early exponential
growth phase (A600 =0.4)
2. Chill on ice and harvest the cells by
centrifugation at 800 g for 20 min
3. Gently resuspend the pellets in 50 mL of
ice-cold 10 % glycerol. Centrifuge as in
step 2
4. Repeat the step 3
5. Repeat the step 3 again
6. Resuspend the cells in a cold 10 % glycerol
at a final concentration of cells 2.5×1010
per milliliter. This suspension may be
frozen in aliquots and stored at −70 °C
Electroporation 1. To a cold, 1.5-mL polypropylene tube, add
50 μL of the cell suspension, 1–4 μL of
BAC DNA (50 nga) in a low ionic strength
buffer such as TE and 1 μg tRNA. Mix
well and let sit on ice for 5 min
2. Set the pulse generator to the 25-μF
capacitor, 2.5 kV, and 300Ω in parallel
with the sample chamber
3. Transfer the mixture of cells and DNA to the
bottom of cold 0.2-cm electroporation cuvette
4. Apply pulse at the above settings. This
should result in a pulse of 12.5 kV/cm with a
time constant of 4.5–5 ms
5. Immediately add 1 mL of LB medium
supplemented with 5 mmol/L MgCl2 at
4 °C to the cuvette and gently but quickly
mix the cells with a pipette
6. Transfer the entire cell suspension from
cuvette to a tube and incubate at 37 °C for
an 1 h with agitation
7. Plate the appropriate aliquots on selective
medium
a
Reduce this amount to 1 ng for transformation with small plasmids
reasonable number. Considering the fact that the sample
resistance is inversely proportional to the volume of solution
in the cuvette, we have chosen the lower value of the sample
volume range recommended by vendor (BioRad). Best trans-
formation efficiency, 1.8×105 cfu/μg DNA, was achieved
with SOB medium lacking magnesium (Fig. 1a). This is not
surprising, since SOB medium is most commonly used for
competent cells preparation and have been already verified
through years of research (Shi et al. 2003). Cells grown in all
other media, LB, 2xYT, and TB (data not shown), resulted in
considerably lower transformation efficiencies in compari-
son with SOB or SOC medium (Fig. 1a). The positive impact
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A B

Transformation efficiency [x 105 cfu/μg DNA]

Transformation efficiency [x 105 cfu/μg DNA]

2.0 18
37°C 21°C
16

1.5 14

12

10
1.0
8

6
0.5
4

2
0.0 0
LB 2xYT SOB- SOB SOC LB 2xYT SOB- SOB SOC- SOC
Media Media

Transformation efficiency [x 105 cfu/μg DNA] C

70
21°C
60

50

40

30

20

10

0
0.2 0.3 0.4 0.5 0.6 0.8 0.9
A 600

D
Transformation efficiency [x 106 cfu/μg DNA]

25
21°C

20

15

10

0
5 10 0.1 0.2 0.3 0.1 0.2 0.3 0.1 0.1 0.2 0.3 0.1 0.2
g/L g/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L mol/L
KAc KAc Su Su Su So So So T M M M X X

E
Transformation efficiency [x 106 cfu/μg DNA]

25
21°C

20

15

10

0
5 10 15 20 0.1 0.3 0.1 0.2 0.3
% % % % mol/L mol/L mol/L mol/L mol/L
H2O Gly Gly Gly Gly T T Su Su Su
 Folia Microbiol
ƒFig. 1 Optimization of electrocompetent DH5α cells preparation. Cells
were grown in different media to an A600 of 0.5 at 37 °C (a) or 21 °C
(b). Presence or absence of Mg and glucose in different media:
SOB-=-Mg/-glucose; SOB=+Mg/-glucose; SOC=+Mg/+glucose;
SOC-=-Mg/+glucose. The detailed composition of media is de-
scribed in the ‘Material and methods’ section. c Transformation
efficiencies of E. coli cells collected at different cell densities
(SOC medium at 21 °C). Influence of various additives (d) and
washing buffers (e) on the transformation rate of cells grown to
A600 of 0.4 at 21 °C in SOC medium. In all experiments, 50 μL
competent cells was transformed in 0.2-cm cuvette with 100 ng of
120 kbp pFML1 plasmid at 12.5 kV/cm, 200-Ω parallel resistor,
and 25-μF capacitance. Error bars indicate the standard deviation.
Su sucrose, So sorbitol, T trehalose, M mannitol, X xylitol, Gly
glycerol
of low-temperature growth (around 20 °C) on transformation
competency is well-established fact, widely used for the
preparation of chemically competent E. coli cells suitable
for high-efficiency transformation (Inoue et al. 1990). Al-
though the same enhancing effect of growth at reduced
temperature on the transformation frequency was already
shown for electrocompetent cells in 1995 (Chuang et al.
1995), most of the protocols, found in the literature and in
the internet, have used 37 °C. The major reason for this is
most likely the significantly prolonged incubation time to
achieve mid-logarithmic phase of growth. We have repeated
the previous experiment with different media at 21 °C. At the
same condition as above, the lowering temperature has min-
imal effect on transformation of DH5α grown in SOB me-
dium without magnesium, but in SOC medium, transforma-
tion efficiency increased from 0.35×105 cfu/mg to 1.65×-
106 cfu/μg of BAC plasmid (Fig. 1b). A SOC medium
without magnesium has shown a similar effect. The above
results indicate that the presence of magnesium in medium
has slightly negative effect on transformation of cells grown
at 37 °C. In contrast, the transformation of cells grown at
21 °C is unaffected by magnesium, and the presence of
glucose seems to be important. It was shown that magnesium
cations could act as positively charged glue between the
negatively charged exterior of the mammalian cells and the
polyanion DNA, forming a complex between the DNA mol-
ecules and the membrane during the application of pulses
(Haberl et al. 2010). On the other hand, excess of magnesium
tightly binds DNA to the membrane and thereby inhibit its
uptake into the cell. Bacterial cell wall constitutes an addi-
tional obstacle to the transfer of DNA into the cell,
preventing the plasmid binding to the plasma membrane.
Therefore, the mechanisms, found in mammalian cells, are
highly unlikely to be involved in electrotransformation of E.
coli. It is known that magnesium stabilizes the membranes,
which leads to increasing of cell viability under stress con-
ditions. Mg2+ is also essential for the production and activity
of extracellular DNase which represent a serious obstacle for
transforming of particular bacteria like Vibrio spp. (Wang
and Griffiths 2009). Extensive washing procedure removes
the major part of magnesium from the surface of the cells;
therefore, it plays only a minor role in electroporation and the
negative effect at 37 °C is only minimal. Significant increas-
ing of the transformation rate of cells grown at low temper-
ature is mainly caused by affecting the physical properties of
the cell membrane such as fluidity due to a change of lipid
composition (Yamanaka 1999). The presence of glucose in
rich medium accelerates the synthesis of glycogen, which
may increase the viability of E. coli under adverse conditions
(Preiss and Romeo 1994). Based on our findings, we have
used for the cultivation of cells in the following experiments
SOC medium at 21 °C. In the most published protocols, E.
coli for competent cells preparation were grown to mid-log
phase (Dower et al. 1988; Miller et al. 1994). To prove the
optimal concentration of cells grown at 21 °C, E. coli DH5α
were harvested at different stages of growth between
A600 =0.2–0.9. As shown in Fig. 1c, cells reached the highest
competency in early logarithmic phase with maximum at
A600 =0.4 (6×106 cfu/μg of BAC plasmid). However, trans-
formation efficiency dramatically decreased above this val-
ue. To enhance the stress tolerance of cells, different addi-
tives have been added to the growth media, e.g., sugars and
salts (Shi et al. 2003). We therefore examined influence of
potassium acetate (KAc), sucrose, sorbitol, trehalose, malt-
ose, and xylitol at different concentrations in the growth
medium on competence of cells. The highest electroporation
efficiency of 1.8×107 cfu/μg of BAC plasmid was observed
in case of SOC medium supplemented with 200 mmol/L
sucrose (Fig.1d). Cells grown in the presence of sorbitol
resulted in a similar transformation rate like control cell
without supplements, all other sugars and KAc were even
less effective than the control (Fig.1d). E.coli in the presence
of KAc required very long cultivation periods, which is
coupled with reduced viability of cells and is probably the
main reason that this additive has proved not to be suitable
for electroporation. We suppose that the enhancement of cell
transformation efficiency of bacteria grown in the presence
of sucrose is caused by increased adaptation of the cell to the
osmotic stress, which is supported by analysis of survival
rates. Fifty-five percent of cells survived electric pulse at
2.5 kV, when were grown in SOC medium, whereas the
presence of 200 mmol/L sucrose in the same medium raised
this number to 75 %.
Preparation of competent cells
It is important to wash the harvested cells thoroughly to
remove all traces of growth media since electroporation in
the presence of high ionic strength causes electrical arcing and
consequently results in the cell death. The most commonly
used washing media are water or 10 % glycerol, the latter of
which minimizes the osmotic shock in porated cells as well as

serves like a cryoprotectant (Miller 1994). Washing solutions
and electroporation buffers can be supplemented with differ-
ent sugars or other nonionic agents to reach suitable osmolar-
ity, which protects the cells in the presence of membrane
pores. Our results demonstrated (Fig. 1e) that 10 % glycerol
yielded the highest competence of cells among the tested
buffers. Water, trehalose, or sucrose were considerably less
effective in all tested concentration. During the washing steps,
we have used mild centrifugation condition, as a consequence
of finding that increasing of centrifugal forces from 800 to
2,000 g caused decreasing of transformation efficiency by a
factor of about 10. This reflects the structural damage of the
cell wall and/or membrane and consequently reduced viability
under stress condition as described previously (Wyber et al.
1994). Further, we have examined the effects of volume used
in the washing procedure. We have used in our preliminary
protocol half of the culture volume of 10 % glycerol. Dou-
bling this volume caused a fourfold increase of transformation
efficiency (7.2×107 cfu/μg of pFML1). This result is in ac-
cordance with the previously observed fact that even traces of
salt decrease transformation rate (Wu et al. 2010) due to
increase of current. Higher current causes sample overheating,
production of more electrochemical product and thereby low-
er survival rate of cells. Consequently, to reach consistent
removal of salt, we have doubled the volume of the washing
buffer in the following experiments.
Considering the importance of knowing competence of
the cells prior to using them for costly transformation such as
library construction, we have examined changes in the trans-
formation efficiency upon freezing. Fresh competent cells
were frozen in liquid nitrogen, transferred to −80 °C freezer
and tested after 1 h, 1 week, 1 month, and 3 months of
storage. The freezing procedure reduced the transformation
efficiency by half compared to the fresh cells, and this value
was stable within the tested period.
Electroporation conditions
As previously described, the optimal field strength decreases
with increasing size of the plasmids (Sheng et al. 1995;
Szostková and Horáková 1998). We therefore examined the
electroporation of large plasmid at different field strengths
ranging from 7.5 to 12.5 kV/cm using competent cells pre-
pared under new conditions. Optimal transformation of E. coli
with BAC plasmid was obtained at electric field 12.5 kV/cm
in 0.2-cm cuvette (7.2×107 cfu/μg of pFML1), which is at the
same time the highest value adjustable on our device (2.5 kV).
To test higher electric field, we repeated this experiment with
0.1-cm cuvettes. The highest transformation efficiency was
achieved at 20 kV/cm, but the transformation was surprisingly
4.8-fold less effective (1.5×107 cfu/μg of pFML1) than at
electric field 12.5 kV/cm in the 0.2-cm cuvette. This effect is
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presumably caused by changes in electric current in different
cuvettes. We suggest that the interplay between DNA concen-
tration, volume, and concentration of cells participate, togeth-
er with electric field geometry, on the final transformation
efficiency and thus these variables have to be optimized for
the each type of cuvette separately. The delivered pulse is
defined by two parameters: the field strength (kV/cm) and
the time constant (s). Duration of capacitor discharge, which is
characterized by the time constant, can be modified by
adjusting of parallel resistor in the circuit. Transformations
were carried out at voltage gradient 12.5 kV/cm in 0.2-cm
cuvette, 25 μF capacitor, 100-, 200-, 300-, 400-, and 700-Ω
resistors and corresponding time constants (averaged) 2.7, 4.7,
6.9, 8.7, and 14.1 ms, respectively (Fig. 2a). The transforma-
tion efficiency showed a broad peak at 200, 300, and 400Ω
with the values very similar to each other (7.2×107, 8.1×107,
and 6.4×107 cfu/μg of pFML1). By using 100- and 700-Ω
resistors the efficiencies were reduced 80-fold and 26-fold,
respectively. The discrepancy to the previously described
optimal resistance of 100Ω for the large plasmids (Sheng
et al. 1995) can be explained by the higher survival rate of
cells growing at low temperature in the presence of 0.2 mol/L
sucrose. Competent cells prepared under these conditions can
be thereby exposed to longer pulses at higher resistances. The
electrical conditions for the electroporation of E. coli have
been verified through years of research; therefore, only minor
modification of established parameters can be found in our
final protocol.
Cell concentration used in electroporation display consid-
erable impact on transformation (Wu et al. 2010). In our
experiment, we have transformed 100 ng pFML1 into the
increasing concentration of the cells, ranging from 1.25 to
7.5×1010 per milliliter (A600 =25–150). The highest transfor-
mation efficiency, 7.2×107 cfu/μg of DNA, was obtained at
our routinely used cell concentration of 2.5×1010 per milliliter
(A600 =50). Interestingly, the transformation yield was dramat-
ically decreased at higher cell densities (Fig. 2b). Our result is
in accordance with previous study (Wu et al. 2010), which
described near identical optimal cell concentration for electro-
poration of DH10B strain. There is not an obvious explanation
for the striking decrease in the transformation rate at higher
cell concentration, but we suppose that the fraction of the
DNA molecules binds in a manner that is inconsistent with
the mechanisms of electrotranslocation through the mem-
brane. Alternatively, the DNA can also bind to dead cells,
which are always present in the competent cells preparations.
In both cases, increased ratio of cell to DNA will decrease the
amount of DNA accessible for transfer into the cells. Trans-
formation efficiency generally increases with quantity of
DNA, but there is a saturation point and the yields can even
decreases with an excess of DNA. In order to examine the
effect of DNA concentration on electrotransformation of E.
Folia Microbiol

A B

Transformation efficiency [x 106 cfu/μg DNA]

Transformation efficiency [x 106 cfu/μg DNA]
100 100

80 80

60 60

40 40

20 20

0 0
100 200 300 400 700 25 50 100 150
Resistance [Ω] A600

C D

Transformation efficiency [x 106 cfu/μg DNA]

Transformation efficiency [x 106 cfu/μg DNA]

140 400

120

100 300

80
200
60

40
100
20

0 0
1 10 25 50 100 200 SOC LB LB + Mg
BAC DNA [ng] Media

Fig. 2 Optimization of electroporation with large BAC plasmid. Influ- rate. Error bars indicate the standard deviation. Details of cell preparation
ence of parallel resistance value (a), cell concentration of competent cells and electrotransformation are described in text (‘Results and discussion’
(b), DNA concentration (c), and recovery media (d) on transformation section)

coli DH5α, we have electroporated 50 μL cell suspension
(concentration of cells 2.5×1010 per milliliter) with a rising
quantity of pFML1 plasmid (Fig. 2c). As expected, number of
transformants increased with the DNA amount, but the trans-
formation efficiency reached the maximum at 50 ng (1.2 ×
108 cfu/μg of pFML1) and slowly decreased beyond this
value (Fig. 2c). The optimal amount of DNA for small plas-
mids usually moves within the 1-ng range (Dower et al. 1988).
Transformation of our competent cell with 1 ng of pBluescript
II KS resulted in 1.5-fold higher efficiency (1.2×1010 cfu/μg)
than with 50 ng of this plasmid (8.2×109 cfu/μg). Considering
the difference in the plasmid sizes and above mentioned DNA
quantities, the optimal molar concentration of both small
(pBluescript) and large (BAC) plasmids is very similar.
This is in line with our finding that particular ratio between
the number of cells and DNA molecules is optimal for the
transformation. Our experiments have revealed that inter-
play between cell concentration and DNA concentration
have significant impact on transformation efficiency and
should be examined carefully for each strain and application,
respectively. We have examinated effect of carrier nucleic acid
on electrotransformation. Our result demonstrates that the
presence of tRNA during the pulse application increases 2.4-
fold the number of clones that can be gained by the transfor-
mation similarly as described previously (Zhu and Dean
1999). The possible mechanism may involve stabilizing the
topological form of the DNA molecules.
The effect of the incubation following the pulse was also
examined. The outgrowth medium was added to the trans-
formation mix immediately after applying the electric pulse.
LB medium (2.0×108 cfu/μg of pFML1) seems to be more
suitable for regeneration than SOC medium (1.2×108 cfu/μg
of pFML1) (Fig. 2d). The addition of MgCl2 to a final
concentration of 5 mM in LB medium further increased the
transformation efficiency by 1.5-fold. Higher or lower
MgCl2 concentrations as well as CaCl2 have no effect or
even decreased the transformation rate (data not shown). As
mentioned above, magnesium stabilizes membranes there-
fore at particular concentration help to seal the pores in lipid
bilayer.
Since the electrotransformation efficiency is strain depen-
dent (Chung et al. 1989; Sheng et al. 1995), we have tested our
protocol for another E. coli strains. Whereas the strains
DH10B is comparable with DH5α in transformation with

the 120 kbp DNA (fivefold reduction), BL21(DE3) and
BW25141 showed a 22- and 59-fold reduction in transforma-
tion efficiency, respectively. To enhance the transformation
rate of the latter two strains, it will be necessary to optimize
each of these strains individually.
Overall, our results have shown that the particular steps in
the competent cells preparation and in electroporation have
contributed to the final transformation efficiency to a differ-
ent extent. Parameters such as the growth temperature, addi-
tion of sucrose into the medium, washing condition, compe-
tent cell, and DNA concentration as well as presence of
tRNA during electroporation displayed the most notable
influence on the transformation. Our optimization experi-
ments resulted in competent cells with transformation effi-
ciency of 7×108 transformants per microgram of large BAC
plasmid. The final protocol is summarized in Table 1. The
competent cells were successfully used for construction of
metagenomic libraries with more than 105 independent
clones (unpublished results). Our competent cells can also
prove valuable for construction of high-complexity cDNA or
genomic libraries with a minimum expenditure of mRNA or
chromosomal DNA. Protocol described here resulted in
competent cells which are comparable with ultra-high-
competency cell from commercial sources and can be valu-
able for low-budget molecular biology laboratory.
Acknowledgments We gratefully acknowledge the financial support
from the Alexander von Humboldt Foundation research group linkage
program and the VEGA (Vedecká Grantová Agentúra Ministerstva
školstva Slovenskej republiky) grant 2/0149/11. We are also thankful
Katarína Kválová for technical assistance.
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