The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

Time courses of cell electroporation as revealed by submicrosecond imaging of transmembrane potential.

Changes in the membrane conductance of sea urchin eggs, during the course of electroporation, were investigated over the time range of 0.5 microsecond to 1 ms by imaging the transmembrane potential at a submicrosecond resolution with the voltage-sensitive fluorescent dye RH292. When a rectangular electric pulse of moderate intensity was applied across an egg, a position-dependent potential developed synchronously with the pulse, as theory predicts for a cell with an insulating membrane. From the rise and fall times, the membrane capacitance of unfertilized eggs was estimated to be 0.95 microF/cm2 and the intracellular conductance 220 omega.cm. Under an electric pulse of much higher intensity, the rise of the induced potential stopped at a certain level and then slowly decreased on the microsecond time scale. This saturation and subsequent reversal of the potential development was ascribed to the introduction of finite membrane conductance, or permeabilization of the membrane, by the action of the intense pulse (electroporation). Detailed analysis indicated the following: already at 0.5 microsecond in the rectangular electric pulse, the two sides of the egg facing the positive and negative electrodes were porated and gave a high membrane conductance in the order of 1 S/cm2; the conductance on the positive side appeared higher. Thereafter, the conductance increased steadily, reaching the order of 10 S/cm2 by 1 ms. This increase was faster on the negative-electrode side; by 1 ms the conductance on the negative side was more than twice that on the positive side. The recovery of the porated membrane after the pulse treatment was assessed from the membrane conductance estimated in a second electric pulse of a small amplitude. At least two recovery processes were distinguished, one with a time constant of 7 microseconds and the other 0.5 ms, at the end of which the membrane conductance was already < 0.1 S/cm2.     

Reversible electrical breakdown of squid giant axon membrane

Charge pulse relaxation experiments were performed on squid giant axon. In the low voltage range, the initial voltage across squid axon membrane was a linear function of the injected charge. For voltages of the order of 1 V this relationship between injected charge and voltage across the membrane changes abruptly. Because of a high conductance state caused by these large electric fields the voltage across the membrane cannot be made large enough to exceed a critical value, Vc, defined as the breakdown voltage, Vc has for squid axon membrane a value of 1.1 V at 12 degrees C. During breakdown the specific membrane conductance exceeds 1 S. cm-2. Electrical breakdown produced by charge pulses of few microseconds duration have no influence on the excitability of the squid axon membrane. The resealing process of the membrane is so fast that a depolarizing breakdown is followed by the falling phase of a normal action potential. Thus, membrane voltages close to Vc open the sodium channels in few microseconds, but do not produce a decrease of the time constant of potassium activation large enough to cause the opening of a significant percentage of channels in a time of about 10 mus. It is probable that the reversible electrical breakdown is mainly caused by mechanical instability produced by electrostriction of the membrane (electrochemical model), but the decrease in the Born energy for ion injection into the membrane, accompanying the decrease in membrane thickness, may play also an important role. Because of the high conductance of the membrane during breakdown it seems very likely that this results in pore formation.     

Studies of cell pellets: II. Osmotic properties, electroporation, and related phenomena: membrane interactions.

Using the relations between pellet structure and electric properties derived from the preceding paper, the responses of rabbit erythrocyte pellets to osmotic or colloidal-osmotic effects from exchanged supernatants and from electroporation were investigated. Changing the ionic strength of the supernatant, or replacing it with dextran or poly(ethylene glycol) solutions, caused changes of Rp according to the osmotic behavior of the pellet. Rp was high and ohmic before electroporation, but dropped abruptly in the first few microseconds once the transmembrane voltage exceeded the membrane breakdown potential. After the initial drop, Rp increased as a result of the reduction of intercellular space. Rp increased regardless of whether the pellets were formed before or immediately after the pulse, indicating that porated cells experienced a slow colloidal-osmotic swelling. The intercellular or intermembrane distances between cells in a pellet, as a function of osmotic, colloidal-osmotic, and centrifugal pressures used to compress rabbit erythrocyte pellets, were deduced from the Rp measurement. This offered a unique opportunity to measure the intermembrane repulsive force in a disordered system including living cells. Electrohemolysis of pelleted cells was reduced because of limited swelling by the compactness of the pellet. Electrofusion was observed when the applied voltage per pellet membrane exceeded the breakdown voltage. The fusion yield was independent of pulse length greater than 10 microseconds, because after the breakdown of membrane resistance, voltage drop across the pellet became insignificant. Replacing the supernatant with poly(ethylene glycol) or dextran solutions, or coating pellets with unporated cell layers reduced the colloidal-osmotic swelling and hemolysis, but also reduced the electrofusion yield. These manipulations can be explored to increase electroloading and electrofusion efficiencies.     

The effects of gramicidin on electroporation of lipid bilayers.

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Cell-attached patch clamp study of the electropermeabilization of amphibian cardiac cells.

Potential gradients imposed across cell or lipid membranes break down the insulating properties of these barriers if an intensity and time-dependent threshold is exceeded. Potential gradients of this magnitude may occur throughout the body, and in particular in cardiac tissue, during clinical defibrillation, ablation, and electrocution trauma. To study the dynamics of membrane electropermeabilization a cell-attached patch clamp technique was used to directly control the potential across membrane patches of single ventricular cells enzymatically isolated from frog (Rana pipiens) hearts. Ramp waveshapes were used to reveal rapid membrane conductance changes that may have otherwise been obscured using rectangular waveshapes. We observed a step increase (delta t less than 30 microseconds) or breakdown in membrane conductance at transmembrane potential thresholds of 0.6-1.1 V in response to 0.1-1.0 kV/s voltage ramps. Conductance kinetics on a sub-millisecond time scale indicate that breakdown is preceded by a period of instability during which the noise and amplitude of the membrane conductance begin to increase. In some cells membrane breakdown was observed to be fully reversible when using an intershock interval of 1 min (20-23 degrees C). These findings support energetic models of membrane electropermeabilization which describe the formation of membrane pores (or growth of existing pores) to a conducting state (instability), followed by a rapid expansion of these pores when the energy barrier for the formation of hydrophilic pores is overcome (breakdown).     

Inactivation of the sodium channel. I. Sodium current experiments

Inactivation of sodium conductance has been studied in squid axons with voltage clamp techniques and with the enzyme pronase which selectively destroys inactivation. Comparison of the sodium current before and after pronase treatment shows a lag of several hundred microseconds in the onset of inactivation after depolarization. This lag can of several hundred microseconds in the onset of inactivation after polarization. This lag can also be demonstrated with double-pulse experiments. When the membrane potential is hyperpolarized to -140 mV before depolarization, both activation and inactivation are delayed. These findings suggest that inactivation occurs only after activation are delayed. These findings suggest that inactivation occurs only after activation; i.e. that the channels must open before they can inactivate. The time constant of inactivation measured with two pulses (τ(c)) is the same as the one measured from the decay of the sodium current during a single pulse (τ(h)). For large depolarizations, steady-state inactivation becomes more incomplete as voltage increases; but it is relatively complete and appears independent of voltage when determined with a two- pulse method. This result confirms the existence of a second open state for Na channels, as proposed by Chandler and Meves (1970. J. Physiol. [Lond.]. 211:653-678). The time constant of recovery from inactivation is voltage dependent and decreases as the membrane potential is made more negative. A model for Na channels is presented which has voltage-dependent transitions between the closed and open states, and a voltage-independent transition between the open and the inactivated state. In this model the voltage dependence of inactivation is a consequence of coupling to the activation process.     

Exponentiated exponential model (Gompertz kinetics) of Na+ and K+ conductance changes in squid giant axon

The conductance changes, gK(t) and gNa(t), of squid giant axon under voltage clamp (Hodgkin and Huxley, 1952) may be modeled by exponentiated exponential functions (Gompertz kinetics) from any holding potential VO to any membrane clamp potential V. The equation constants are set by the membrane potential V, and include, for any voltage step in the case of gK, the initial conductance, gO, the asymptote conductance g, and rate constant k: gK = g exp(-be-kt) where b = 1n g/gO. Equations of similar form relate g and k to the voltage V, and govern the corresponding parameters of the gNa system. For the gNa, the fast phase y = y exp (-be-kt) is cut down in proportion to a slow process p = (1 - p)e-k't + p, and thus gNa = py. The expo-exponential functions involve fewer constants than the Hodgkin-Huxley model. In particular, the role of the n, m, h parameters appears to be filled largely by 1n (g/gO) in the case of gK and by 1n (y/yO) in the case of gNa. Membrane action potentials during current clamp may be computed from the conductances generated by use of the appropriate differential forms of the equations; diverse other membrane behaviors may be predicted.     

Biphasic voltage relaxation pattern observed in cells of Eremosphaera viridis after injection of charge-pulses of short duration: detection of tip clogging of intracellular microelectrodes by charge-pulse technique

Charge pulse experiments performed on the peat-bog alga Eremosphaera viridis revealed an unusual voltage relaxation behaviour. Injection of charge pulses of 1 microseconds duration resulted in an immediate charging of the membranes (time constant of the order of 40 ns). Nevertheless, the potential-measuring microelectrode recorded an exponential increase in membrane voltage with a time constant of about 1.3 ms. The maximum voltage value was recorded after about 3 ms, followed by an exponential decay with a time constant of about 9.6 ms. This biphasic time course was independent of the amplitude of the injected charge and of the location of the impaled microelectrodes in the vacuole. Centrifuged cells in which the chloroplasts and the other organelles were pelleted in one part of the cells showed the same electrical response. Electrical breakdown of the cell membranes resulted in the disappearance of the biphasic voltage response. In this case only the decaying relaxation process could be recorded with a time constant of 3 ms. After resealing of the membranes the original biphasic relaxation response was restored. Increasing concentrations of KCl in the bathing medium reduced both time constants almost correspondingly. The experimental findings were evaluated with an electrical equivalent circuit. Theoretical analysis with reference to the experimental data suggested that the delayed voltage response of the potential-recording electrode resulted from a membrane seal across the tip of this electrode. The resistance of this seal was calculated to be about 400 M omega. The specific resistances and capacitances of tonoplast and plasmalemma membranes were calculated from the decaying part of the biphasic relaxation curves. The average values were found to be 2.58 omega.m2 and 5 mF.m-2. The investigations reported here suggest that charge pulse experiments can be generally used for the detection of membrane and cytoplasmic material clogging of the tip of intracellular microelectrodes, a problem with which most electrophysiologists are faced when interpreting data obtained from impaled microelectrodes.     

Breakdown of lipid bilayer membranes in an electric field

The behaviour of lipid bilayer membranes, made of oxidized cholesterol, and UO₂²⁺-modified azolectin membranes in a high electric field has been investigated using the voltage clamp method. When a voltage pulse is applied to the membrane of these compositions, the mechanical rupture of the membranes is preceded by a gradual conductance increase which remains quite reversible till a certain moment. The voltage drop at this reversible stage of breakdown leads to a very rapid (characteristic time of less than 5 μs) decrease in the membrane conductance. At repeated voltage pulses of the same amplitude with sufficient intervals between them (approx. 10 s), the current oscillograms reflecting the reversible resistance decrease are well reproduced on the same membrane. The time of attainment of the predetermined level of the membrane conductance is strongly dependent on voltage. At different stages of breakdown we have investigated changes in the conductance of UO₂²⁺-modified membrane after the application of two-step voltage pulses, the kinetics of development of the reversible decrease in the membrane resistance in solutions of univalent and divalent ions, and also the influence of sucrose and hemoglobin on the current evolution. The relationship between the reversible conductance increase, the reversible electrical breakdown [15] and the rupture of membrane in an electric field is discussed. We propose the general interpretation of these phenomena, based on the representation of the potential-dependent appearance in the membrane of pores, the development of which is promoted by an electric field.     

Supercharging: a method for improving patch-clamp performance.

Patch-clamp performance can be improved without altering the normal headstage configuration described by (Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth, 1981, Pfluegers Arch. Eur. J. Physiol., 391:85-100). The "supercharging" method permits resolution of such fast events as calcium and sodium tail currents. Digital computer modeling and analog electronic simulation were used to identify appropriate shapes for the command voltage and the voltage applied to a capacitor tied to the input of the headstage. The voltage command pulse consists of a step with a brief (5-15 microseconds) rectangular spike on its leading edge. Spike amplitude is a function of the membrane capacitance and the access resistance. The spike drives current through the access resistance and speeds charging of the membrane capacitance, making it possible to complete a voltage step within 5-15 microseconds. Clamping speed is independent of the electrode and feedback resistance over a wide range. The second function of the patch clamp amplifier is current measurement, and good time resolution requires suppression of the capacity transient. This can be accomplished by applying an appropriately shaped voltage to the small capacitor tied to the input of the headstage. Series resistance compensation for ionic current transients does not interfere with supercharging. Although the focus of this paper is on whole cell recording, the supercharging concept may prove useful for single channel and bilayer recording techniques.     

Electroporation: A general phenomenon for manipulating cells and tissues

Electroporation is a fascinating cell membrane phenomenon with several existing biological applications and others likely. Although DNA introduction is the most common use, electroporation of isolated cells has also been used for (1) introduction of enzymes, antibodies, and other biochemical reagents for intracellular assays; (2) selective biochemical loading of one size cell in the presence of many smaller cells; (3) introduction of virus and other particles; (4) cell killing under nontoxic conditions; and (5) insertion of membrane macromolecules into the cell membrane. More recently, tissue electroporation has begun to be explored, with potential applications including (1) enhanced cancer tumor chemotherapy, (2) gene therapy, (3) transdermal drug delivery, and (4) noninvasive sampling for biochemical measurement. As presently understood, electroporation is an essentially universal membrane phenomenon that occurs in cell and artificial planar bilayer membranes. For short pulses (μs to ms), electroporation occurs if the transmembrane voltage, U(t), reaches 0.5–1.5 V. In the case of isolated cells, the pulse magnitude is 10³–10⁴ V/cm. These pulses cause reversible electrical breakdown (REB), accompanied by a tremendous increase molecular transport across the membrane. REB results in a rapid membrane discharge, with the elevated U(t) returning to low values within a few microseconds of the pulse. However, membrane recovery can be orders of magnitude slower. An associated cell stress commonly occurs, probably because of chemical influxes and effluxes leading to chemical imbalances, which also contribute to eventual survival or death. Basic phenomena, present understanding of mechanism, and the existing and potential applications are briefly reviewed.     

Tissue electroporation. Observation of reversible electrical breakdown in viable frog skin.

Experiments by others have used isolated cell or bilayer membrane preparations to study the dramatic phenomena associated with electroporation. The present study observes electroporation behavior in an intact tissue. Viable samples of frog skin (Rana pipiens) were exposed to short electrical pulses of varying width and magnitude under "charge injection" conditions. After a pulse, the transtissue potential decayed with two distinct time constants, one short (tau approximately 0.3 ms) and the other longer (tau L approximately 2 ms). Above thresholds for the pulse magnitude and for the pulse width tau L decreased significantly, with progressively smaller tau L as the pulse magnitude and width increased. The postpulse potential, delta Utissue (t), and resistance, Rtissue, also decreased progressively. The tissue subsequently recovered to its original resistance and open circuit potential, delta U tissue,oc, within 2-3 min after a pulse. At that time another pulse experiment could be carried out, demonstrating repeatability and reversibility. No significant permanent changes in Rtissue and delta Utissue,oc were found. This is interpreted as avoidance of significant tissue damage. Taken together, these dramatic phenomena are characteristic of the reversible electrical breakdown previously observed in charge injection experiments with artificial planar bilayer membranes and with isolated cell membranes by similar very short pulses. The present experiments therefore demonstrate that electroporation can be repeatedly caused and observed in a viable tissue without apparent damage.     

Electroporation of cell membrane visualized under a pulsed-laser fluorescence microscope.

Controlled permeability can be conferred to cell membranes by exposing cells to a microsecond electric pulse of sufficient intensity (electroporation). By constructing a fluorescence microimaging system with a submicrosecond time resolution we have been able to resolve temporally and spatially the events in a single cell under a microsecond electric pulse. An enormous membrane conductance, corresponding to a loss of 0.01-0.1% of the membrane area, was observed in those membrane regions where the transmembrane potential induced by the electric pulse exceeded a critical value. The conductance decreased to a low level in a submillisecond after the pulse, leaving a moderately electroporated cell.     

Membrane conductance of an electroporated cell analyzed by submicrosecond imaging of transmembrane potential.

Transmembrane potential was induced in a sea urchin egg by applying a microsecond electric pulse across the cell. The potential was imaged at a submicrosecond time resolution by staining the cell membrane with the voltage-sensitive fluorescent dye RH292. Under moderate electric fields, the spatial distribution of the induced potential as well as its time dependence were in accord with the theoretical prediction in which the cell membrane was regarded as an insulator. At higher field intensities, however, the potential apparently did not fully develop and tended to saturate above a certain level. The saturation is ascribed to the introduction of a large electrical conductance, in the form of aqueous openings, in the membrane by the action of the induced potential (electroporation). Comparison of the experimental and theoretical potential profiles indicates that the two regions of the membrane that opposed the electrodes acquired a high membrane conductance of the order of 1 S/cm2 within 2 microseconds from the onset of the external field. The conductance was similar in the two regions, although permeability in the two regions of the membrane long after the pulse treatment appeared quite different.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

High electric field effects on the cell membranes of Halicystis parvula

The electrical breakdown behavior of the giant algal cell Halicystis parvula was studied in order to predict the optimum conditions for electrically induced cell-to-cell fusion. Using the charge pulse technique, the membranes were charged at different pulse lengths to the maximum voltage V_c. Because of a reversible, high-conductance state of the membrane (electrical breakdown), it was not possible to exceed the critical membrane breakdown potential. The breakdown voltage exhibited a strong dependence on the charging time (pulse length) between 10 s and 100 s. Below 10 s the breakdown voltage of the two membranes, tonoplast and plasmalemma, assumed a constant value of about 1.9 V, whereas above a pulse length of about 100 s the breakdown voltage was nearly constant with a value of about 0.6 V. The extreme values for the breakdown voltage at very short and at very long charging times agree fairly well with results which have been obtained on cells of Valonia utricularis and planar lipid bilayer membranes. However, the pulse length dependence of the breakdown voltage was found to be quite different in H. parvula. In addition, the membrane conductance increase during breakdown in H. parvula cells is much more pronounced than in membranes of V. utricularis, but similar to lipid bilayer membranes. From this result it is suggested that the membrane structure of H. parvula is quite different from V. utricularis (larger lipid domains).     

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation.

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.     

Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation

To apply recombinant DNA techniques for genetic manipulation of the industrially important lactococci, an efficient and reliable high-frequency transformation system must be available. High-voltage electric pulses have been demonstrated to enhance uptake of DNA into protoplasts and intact cells of numerous gram-negative and gram-positive microorganisms. The objective of this study was to develop a system for electroporating intact cells of Lactococcus lactis subsp. lactis LM0230 (previously designated Streptococcus lactis LM0230) with a commercially available electroporation unit (BTX Transfector 100; BTX, Inc., San Diego, Calif.). Parameters which influenced the efficiency of transformation included growth phase and final concentration of cells, ionic strength of the suspending medium, concentration of plasmid DNA, and the amplitude and duration of the pulse. Washed suspensions of intact cells suspended in deionized distilled water were subjected to one high-voltage electric pulse varying in voltage (300 to 900 V corresponding to field strengths of 5 to 17 kV/cm) and duration (100 microseconds to 1 s). Transformation efficiencies of 10(3) transformants per microgram of DNA were obtained when dense suspensions (final concentration, 5 x 10(10) CFU/ml) of stationary-phase cells were subjected to one pulse with a peak voltage of 900 V (field strength, 17 kV/cm) and a pulse duration of 5 ms in the presence of plasmid DNA. Dilution of porated cells in broth medium followed by an expression period of 2 h at 30 degrees C was beneficial in enhancing transformation efficiencies. Plasmids ranging in size from 9.8 to 30.0 kilobase pairs could be transformed by this procedure.