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Optimization of E. coli culture conditions for
efficient DNA uptake by electroporation
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Introduction
 Bacterial transformation is a good breakthrough in the field of
molecular biology for cloning purposes
 For carrying out transformation the basic requirements are
Foreign DNA
Vector
Cells having capability to take up exogenous DNA
Transformation technique
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Further transformation results could be checked out
initially by
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Antibiotic selection
Blue/white screening
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Blue/white screening(Bacterial colonies
transformed with pUC18)
White colonies
(contain recombinant DNA
molecules)
blue colonies
(contain non-recombinant
DNA molecules)
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How it will be confirmed that either your transformation experiment is
successful or not
 Restriction digestion
 PCR amplifications
 Sequencing
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Gene transfer results can be stable or transient
 Transient :
 DNA is not integrated into host chromosome. DNA is transferred
into recipient cells in order to obtain a temporary but high level of
expression of target gene
 Stable:
 The transferred DNA is integrated into chromosomal DNA and the
genetics of cell is permanently changed
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 Thus for a bacterial cell to take up DNA from its
surroundings, it must be in a special physiological state
called competence
 Competency is the ability of bacterial cells to take up
exogenous DNA from close vicinity
OR
 Competent cells are those that possess more easily altered
cell walls that DNA can be passed through easily
 Competence is distinguished into
 Natural competence
 Artifical Competence
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Natural competence
 Bacteria are able to take up DNA from their environment
(exogenous DNA) in three ways;
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Conjugation
Transformation
Transduction
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 Natural competence is highly regulated in bacteria, and the factors leading
to competence vary among genera
 For some genera, only a portion of the population is competent at any time; for
others, the entire population gains competence
 A series of competence proteins is produced, which have some homology but
differ in the Gram negative and the Gram positive bacteria.
 Once the DNA has been brought into the cell's cytoplasm
 It may be degraded by cellular nucleases
 If it is very similar to the cells own DNA, enzymes that normally repair DNA
may recombine it with the chromosome.
 Transformation can occur naturally in
 Streptococcus, Bacillus Acinetobacter, Neisseria, and Pseudomonas
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Artifical competence
 There are two methods to induce artificial competence
 Chemical
 Physical
 Chemically induced competence followed by transformation is
a commonly used technique to introduce plasmids or other
DNA fragments into Escherichia coli.
 In chemical method bivalent cations (Ca+2, Mg+2, Ba+2, Rb+
and Sr+2) are chiefly used to induce competence in cells
(Huang and Reusch, 1995)
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Contd…
 Beside these many other chemicals e.g.
 EDTA (Williams et al., 1996),
 Polyethylene glycol (Himeno et al., 1984)
 Osmotic agents including
 Sucrose (Antonov et al., 1993)
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Glycine (Holo and Nes, 1989)
Sorbitol and manitol (Xue et al., 1999)
 These
chemicals not only increase the chances of
transformation but it also improves the chances of cell survival
when present in high concentrations in the electroporation
medium
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 Alteration in the permeability of the membranes allows DNA to
cross the cell envelope of E. coli which is composed of an outer
membrane, an inner membrane, and a cell wall.
 The outer membrane of E. coli can be understood by
application of the fluid mosaic model for membranes and is
composed of phospholipids, proteins, and lipopolysaccharides
 Many channels or zones of adhesions are formed by the fusion
of the outer membrane and the inner membrane through the
cell wall layer
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 Although the transformation mechanism is not known,
previous studies indicate that
 The channels between outer and inner membrane allow for the transport
of DNA molecules across the cell membrane.
 The negative charges of the incoming DNA, however are repelled by the
negatively charged portions of the macromolecules on the bacterium’s
outer surface.
 The addition of CaCl2 serves to neutralize the unfavorable interactions
between the DNA and the polyanions of the outer layer
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 The DNA and competent cells are further incubated on
ice for thirty minutes to stabilize the lipid membrane and
allow for increased interactions between calcium ions
and the negative components of the cell
 The reaction mixture is then exposed to a brief period of
heat-shock at 42oC
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Alters fluidity of membranes
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Methods to induce artifical
competence
 There are two methods to induce artificial competence
 Chemical
 Physical
 Electroporation is a most popular physical process of
producing transient pores in outer membrane of living cells by
subjecting them to a rapidly changing high strength electric
field.
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Contd..
 Electroporation is enhanced by several factors, Wu et al. (2010)
reported that careful control of
(i) Cell growth phase
(ii) Electroporation cell number
(iii) Cell desalination with glycerol
(iv) Amount of transforming DNA may enhance transformation efficiency
of E. coli up to 1010 transformants/µg DNA (10-40pg)
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Reasons for preference of physical method
over chemical method of competent cell
preparation.
 Method of preparing electrocompetent cells is preferred over
the chemical method for transferring exogenous DNA into
bacteria, because of its
 High transformation efficiency that is suitable for regular plasmid,
ligated cDNA, and genomic DNA transformation
 Suitability
of using less transforming DNA amount for high
transformation efficiency, which is beneficial for rare and/or hard-toobtain gene DNA
 Transformation irreversibility compared to chemical transformation
method (Kurien and Scofield, 1995).
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 Limitations
 During conventional electrocompetent cell preparation, extra-sodium
chloride is normally used as a major ion source in the medium (about
1% in LB medium).
 The combination of chemical-chemical, chemical-physical and physical-
physical methods improves the transformation manifolds and is used in
laboratories worldwide.
 Bukau et al. (1985) suggested that the growth medium supplemented with
sucrose facilitates DNA uptake
 Transformation efficiency of E. coli may also be enhanced by the use of
simplest amino acid, Glycine (1% w/v) in growth medium reported by
Akhtar et al. (1999).
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 Ahmed et al. 2014 utilize LB and SOC media and their
supplemented combinations (Sucorse and Glycine) to prepare
electro-competent cells of E. coli DH5α at temperature 25 and
37 °C which were then subjected to electroporation.
 For carrying out their study they utilize
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DH5 α strain of E. coli
The plasmid pENTRTM/D-TOPO® gateway entry vector of size 2580
bp was used to produce recombinant vector pENTR/D-TOPO-RGLP1
of 3255 bp by ligation of 675 bp long fragment.
The vector harbors kanamycin resistance gene as a selection marker.
Media combinations (LB, LB-S, LB-SG, LB-G) (SOC, SOC-S, SOCSG, SOC-G)
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 What methodology they adopted
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Growth kinetics of E. coli DH5α was studied in all the combinations
of media at temperatures 37 ºC and 25 ºC
Electrocompetent cells of E. coli DH5α was prepared by following
protocol of Sambrook and Russel (2001) with certain modifications,
In this study competent cells were prepared in media combinations of
LB, LB-S, LB-SG, LB-G, SOC, SOC-S, SOC-SG, SOC-G at two
different temperatures 25 ºC and 37 ºC
Electroporation of competent cells
PCR confirmation of plasmid
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 The results showed that cells grown in SOC media showed
high growth rates and increased transformation efficiencies as
compared to LB.
 Results of growth kinetics show that SOC-S and SOC-SG are
better media combinations for achieving required density of
0.3- 0.45 for preparing competent cells at both temperatures in
less time as evident from the comparison of results depicted in
Figures 1, 2, 3 and 4.
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 However efficient growth kinetics of E. coli is not an indication
that it will transform efficiently as it has been reported by Wu
et al. (2010) that transformation efficiency is inversely
proportional to cell population size
 The increased growth rate in presence of SOC media might be
due to the fact that SOC is a glucose containing nutrient rich
media, and glucose is a readily metabolizable sugar (Tu et al.,
2005)
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 In contrast, glycine when added to growth media may interfere
with the biosynthesis of membranes by replacing L- and Dalanine from peptidoglycan with glycine and thus reducing cell
growth (Kaderbhai et al., 1997)
 As glycine is metabolized into acetate while acetate is an
inhibitor of bacterial growth in this way it reduces growth rate
of E. coli (Luli and Strohl, 1990).
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 Further
increased transformation efficiencies were
observed by Ahmed et al. 2014 in SOC media as compared
to LB media which has been speculated to be due to
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Presence of bivalent cation (MgCl2) in SOC media which helps to
facilitate DNA uptake
 In
all combinations tested highest transformation
efficiencies were observed for the cells grown in SOC-SG at
25 ºC i.e. 3.56 × 109 cfu/µg of DNA might be due to
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Mass flow of sucrose along with transformation mixture
Structural impariments
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 In addition to media combinations of osmotic agents
temperature, was also tested as parameter for enhancing
electroporation efficiency
 An ehanced electroporation rate at lower temperature (25 ºC)
as compared to optimum temperature for DH5α growth as
demonstrated in Table 2
 When cells are grown in temperature lower then optimum temperature
(37 ºC), proportion of unsaturated fatty acids in their membranes
increases (Gill and Suisted, 1978) which increase fluidity and can
interfere membrane functions (Kuo et al., 1990) thus making
membrane more permeable to DNA.
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Conclusions
 This study supports previous findings that osmotic agents and lower
temperature are imperative factors in enhancing DNA uptake efficiency
 Ahmed et al. 2014 concluded that efficient culture media for increasing
number of transformants per µg DNA is SOC media, supplemented with
Sucrose and glycine as compared to LB.
 The investigation reinforces the notion that even limited size/ low budget
molecular biology laboratories could efficiently prepare highly efficient
electrocompetent cells with transformation efficiencies of 3.56 x
109cfu/µgDNA.
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Reference
 Irfan
Ahmed, Tehseen Rubbab*, Farah Deeba, Syed
Muhammad Saqlan Naqvi. Optimization of E. coli culture
conditions for efficient DNA uptake by electroporation. Turk J
Biol. (2014), 38:568-573.
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Thanks 
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