Biomedical Engineering Reference
In-Depth Information
addition to calcium, have been shown to stimulate
the process.
A very simple, moderately efficient transforma-
tion procedure for use with
E. coli
involves resus-
pending log-phase cells in ice-cold 50 mmol/l
calcium chloride at about 10
10
cells/ml and keeping
them on ice for about 30 min. Plasmid DNA (0. 1
sources (see Chapter 3) and reduce the transforma-
tion efficiency. Large DNAs transform less effici-
ently, on a molar basis, than small DNAs. Even with
such improved transformation procedures, certain
potential gene-cloning experiments requiring large
numbers of clones are not reliable. One approach
which can be used to circumvent the problem of low
transformation efficiencies is to package recombin-
ant DNA into virus particles
in vitro
. A particular
form of this approach, the use of cosmids, is described
in detail in Chapter 5. Another approach is electro-
poration, which is described below.
g)
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. (This so-called
phenotypic lag
may not need to be taken into consideration with
high-level ampicillin resistance. With this marker,
significant resistance builds up very rapidly, and
ampicillin exerts its effect on cell-wall biosynthesis
only in cells which have progressed into active
growth.) Finally the cells are plated out on selective
medium. Just why such a transformation procedure
is effective is not fully understood (Huang & Reusch
1995). 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.
Hanahan (1983) has re-examined factors that
affect the efficiency of transformation, and has devised
a set of conditions for optimal efficiency (expressed
as transformants per
µ
Electroporation
A rapid and simple technique for introducing cloned
genes into a wide variety of microbial, plant and ani-
mal cells, including
E. coli
, is electroporation. This
technique depends on the original observation by
Zimmerman & Vienken (1983) that high-voltage
electric pulses can induce cell plasma membranes to
fuse. Subsequently it was found that, when sub-
jected to electric shock, the cells take up exogenous
DNA from the suspending solution. A proportion of
these cells become stably transformed and can be
selected if a suitable marker gene is carried on the
transforming DNA. Many different factors affect the
efficiency of electroporation, including temperature,
various electric-field parameters (voltage, resistance
and capacitance), topological form of the DNA,
and various host-cell factors (genetic background,
growth conditions and post-pulse treatment). Some
of these factors have been reviewed by Hanahan
et al
. (1991).
With
E. coli
, electroporation has been found to give
plasmid transformation efficiencies (10
9
cfu/
g plasmid DNA) applicable to
most
E. coli
K12 strains. Typically, efficiencies of 10
7
to 10
9
transformants/
µ
g can be achieved depending
on the strain of
E. coli
and the method used (Liu &
Rashidbaigi 1990). Ideally, one wishes to make a
large batch of competent cells and store them frozen
for future use. Unfortunately, competent cells made
by the Hanahan procedure rapidly lose their com-
petence on storage. Inoue
et al
. (1990) have optimized
the conditions for the preparation of competent cells.
Not only could they store cells for up to 40 days at
−
µ
g DNA)
comparable with the best CaC1
2
methods (Dower
et al
.
1988). More recently, Zhu and Dean (1999) have
reported 10-fold higher transformation efficiencies
with plasmids (9
µ
10
9
g) by co-
precipitating the DNA with transfer RNA (tRNA)
prior to electroporation. With conventional CaCl
2
-
mediated transformation, the efficiency falls off
rapidly as the size of the DNA molecule increases
and is almost negligible when the size exceeds 50 kb.
While size also affects the efficiency of electroporation
(Sheng
et al
. 1995), it is possible to get transforma-
tion efficiencies of 10
6
×
transformants/
µ
10
9
cfu/
g,
but competence was affected only minimally by salts
in the DNA preparation.
There are many enzymic activities in
E. coli
which
can destroy incoming DNA from non-homologous
70°C while retaining efficiencies of 1-5
×
µ
cfu/
µ
g DNA with molecules
