Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 54)

Section 5
Factors Affecting Electroporation: Optimizing Electroporation
The electrical conditions for the electroporation of cells have been verified through years of research (see
Shigekawa & Dower, 1988, Change, et al., 1992, and Nickoloff, 1995a,b, for overviews as well as for
protocols on electroporation of numerous species). For suggestions on optimizing electroporation
parameters, see the following references: Calvin & Hanawalt, 1988, Dower, et al., 1988 (for bacterial
cells); Shillito, et al., 1985, Fromm, et al., 1987, Dekeyser, et al., 1990, Joersbo & Brunstedt, 1991 (for
plant cells); Chu, et al., 1987, Knutson & Yee, 1987, Andreason & Evans, 1989, Anderson, et al., 1989,
and Heiser, 1999 (for mammalian cells). For microorganisms, optimum electrotransformation occurs
using exponential decay pulses under electrical conditions similar to those used for E. coli and S. cerevisiae,
two species that are most commonly used in research today. The pulse conditions consist of a low
capacitance (short pulse duration) at high voltage. For E. coli, conditions that result in a field strength of
~18 kV/cm and a time constant of ~5 msec are optimal. For many bacterial species, including
Salmonella, Borrelia, Lactococcus, and Enterococcus, the conditions for electroporation are generally
identical to those used for E. coli. For many other bacterial species, altering the field strength will often
result in higher electrotransformation. For S. cerevisiae, conditions that result in a field strength of
~7.5 kV/cm and a time constant of ~5 msec are near optimal. Similar optimal conditions are also found
with other species of yeast.
Successful electroporation of plant protoplasts has been obtained using exponential decay pulses at
both low-capacitance and high-voltage settings or high-capacitance (long pulse duration) and low-voltage
settings. Typical conditions are time constants of 10–100 µsec and field strengths of 2.5 –5 kV/cm or
time constants of 2–5 msec and a field strength of about 500 V/cm.
For mammalian cells, optimum electrotransformation conditions have been reported using both
exponential decay and square wave pulses. Typical conditions for both types of pulses employ
pulse lengths or time constants of 10–40 msec and field strengths of 400–900 V/cm. Generally
there is an inverse relationship between field strength and pulse length or time constant such that,
over a limited range, one variable may be increased and the other decreased in order to maintain
the optimum electroporation conditions. Additionally, as cell size increases, the field strength at
which optimum electrotransformation occurs generally decreases. Other conditions that are
important for maximizing electroporation efficiency are discussed in the following sections.
5.1 Cell growth
The optimal portion of the growth phase to harvest cells is generally dependent on the cell type. When
preparing competent cells of a new species it is generally best to start with conditions employed for use within
the same genus. Suggestions for factors to consider and general methods for producing electrocompetent
cells are discussed in the articles by Dower, et al. (1992) and Trevors, et al. (1992). For most bacterial species,
the highest transformation efficiencies are obtained when cells are harvested in early to mid-log growth. With
E.coli, as the cells reach stationary phase, the transformation efficiency declines precipitously (Dower, 1990). In
contrast, most yeast species are generally harvested in mid- to late-log growth. For S. cerevisiae, the
transformation efficiency increases as much as 60-fold from early to late-log cultures (Becker and Guarente,
1991). For mammalian cells, the highest expression following electroporation is obtained when cells are in
mid-log phase growth (Anderson, et al., 1991).
48