High efficiency transformation of E. coli by high voltage electroporation.

E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.     

Optimization of electrotransformation conditions for Propionibacterium acnes

Propionibacterium acnes has been known to be involved in the pathology of acne. However, the definite mechanism in the development of acne and the inflammation are unknown. For P. acnes, a transformation method has not been established, although it is believed to be a basic tool for gene manipulation. This study attempted to develop a P. acnes transformation method by using electroporation. Various parameters were used to develop and optimize the transformation of P. acnes. Among them two factors were crucial in the transformation for P. acnes: one was the E. coli strain from which the plasmid DNA had been isolated and the other the growth temperature of P. acnes-competent cells. It was essential to prepare plasmid DNA from a dam(-) E. coli strain, ET12567. When plasmid DNAs isolated from the other E. coli strains such as JM109 and HB101 were tested, transformation efficiency was extremely low. When P. acnes cells were cultivated at 24 degrees C for competent cell preparation, transformation efficiency increased considerably. When plasmid DNA isolated from a dam(-) mutant strain of E. coli was used for transformation of P. acnes which had been grown at 24 degrees C, maximum transformation efficiency of 1.5 x 10(4) transformants per mug of plasmid DNA was obtained at a field strength of 15 kV/cm with a pulse time of 3.2 ms. This is believed to be the first report on the transformation of P. acnes which can be employed for gene manipulations including knock-out of specific genes.     

DNA transfection of Escherichia coli by electroporation

Electroporation was applied to transfection and transformation of Escherichia coli. Efficient transfer of DNA was achieved by a single voltage pulse at 2.5 kV (initial electric field strength = 6.25 kV/cm), with a 25 microF capacitor. As the recipient for transfecting DNA in the electroporation, spheroplasts, EDTA-treated cells and osmotically shocked bacteria were inferior to intact E. coli. Various parameters affecting the transfection efficiency were defined including growth phase of recipient cells, concentrations of DNA and cells, temperature and additions. In most strains tested, electroporation was far more efficient than Ca2+-dependent transfection (transformation). Various aspects of the electroporation-mediated DNA uptake are discussed.     

Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation

An efficient method for the transformation of the methylotrophic bacterium Methylobacterium extorquens NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of M. extorquens NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from Escherichia coli. Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10³ transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.     

A hierarchical procedure for forming informative features of a pulse signal applied to the problem of identification of early signs of hypertension in childhood and adolescence

The suggested approach for searching informative features of a pulse signal is based on sequential analysis of the characteristics of the waveform and the shape of rhythmic structure of the signal. At the first stage of the analysis, the pulse waveform is determined. The results allow the algorithm of the analysis of the rhythmic structure to be selected depending on the type of the pulse waveform. The difference of the analysis algorithms is caused by the fact that the type of the waveform determines the number of local parameters that affect the rhythmic structure. The suggested algorithm allows its parameters to be corrected at each stage of the analysis depending on the results of the previous stage. This gives the opportunity to take specific features of the pulse signal into account and, hence, improves the quality of recognition of diseases by the pulse analysis.     

Electro-stimulated transformation of E. coli cells pretreated by EDTA solution

The phenomenon of transformation of E. coli cells under electric treatment has been studied. The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension. The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA. The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.     

Rapid electroporation-mediated plasmid transfer between Lactococcus lactis and Escherichia coli without the need for plasmid preparation

A simple and rapid method for the transfer of plasmids between the Gram-positive species Lactococcus lactis and Escherichia coli without the need for plasmid preparation is described. The donor strain was subjected to an electroporation pulse which released plasmid DNA into the suspending buffer which was then centrifuged to remove cells and debris. The supernatant was mixed with the recipient strain and subjected to a second electroportion pulse, resulting in the transfer of plasmid from donor to recipient. In cases where a high transformation efficiency is not required, such as the transfer of a cloned construct from E. coli to Lactococcus or vice versa , this method has the advantages of convenience and rapidity.     

Effect of non-symmetric waveform on conduction block induced by high-frequency (kHz) biphasic stimulation in unmyelinated axon

The effect of a non-symmetric waveform on nerve conduction block induced by high-frequency biphasic stimulation is investigated using a lumped circuit model of the unmyelinated axon based on Hodgkin-Huxley equations. The simulation results reveal that the block threshold monotonically increases with the stimulation frequency for the symmetric stimulation waveform. However, a non-monotonic relationship between block threshold and stimulation frequency is observed when the stimulation waveform is non-symmetric. Constant activation of potassium channels by the high-frequency stimulation results in the increase of block threshold with increasing frequency. The non-symmetric waveform with a positive pulse 0.4–0.8 μs longer than the negative pulse blocks axonal conduction by hyperpolarizing the membrane and causes a decrease in block threshold as the frequency increases above 12–16 kHz. On the other hand, the non-symmetric waveform with a negative pulse 0.4–0.8 μs longer than the positive pulse blocks axonal conduction by depolarizing the membrane and causes a decrease in block threshold as the frequency increases above 40–53 kHz. This simulation study is important for understanding the potential mechanisms underlying the nerve block observed in animal studies, and may also help to design new animal experiments to further improve the nerve block method for clinical applications.     

Analysis and Optimization of Pulse Dynamics for Magnetic Stimulation

Magnetic stimulation is a standard tool in brain research and has found important clinical applications in neurology, psychiatry, and rehabilitation. Whereas coil designs and the spatial field properties have been intensively studied in the literature, the temporal dynamics of the field has received less attention. Typically, the magnetic field waveform is determined by available device circuit topologies rather than by consideration of what is optimal for neural stimulation. This paper analyzes and optimizes the waveform dynamics using a nonlinear model of a mammalian axon. The optimization objective was to minimize the pulse energy loss. The energy loss drives power consumption and heating, which are the dominating limitations of magnetic stimulation. The optimization approach is based on a hybrid global-local method. Different coordinate systems for describing the continuous waveforms in a limited parameter space are defined for numerical stability. The optimization results suggest that there are waveforms with substantially higher efficiency than that of traditional pulse shapes. One class of optimal pulses is analyzed further. Although the coil voltage profile of these waveforms is almost rectangular, the corresponding current shape presents distinctive characteristics, such as a slow low-amplitude first phase which precedes the main pulse and reduces the losses. Representatives of this class of waveforms corresponding to different maximum voltages are linked by a nonlinear transformation. The main phase, however, scales with time only. As with conventional magnetic stimulation pulses, briefer pulses result in lower energy loss but require higher coil voltage than longer pulses.     

An evaluation of some electrical waveforms and voltages used for electric fishing; with special reference to their use in backpack electric fishing gear

Exponential pulse waveform in electric fishing gave a significantly poorer capture efficiency than other waveforms evaluated. Plasma cortisol levels were significantly and uniformly elevated after shocking by all waveforms. Mortality from the electric fishing was low, 0·2% overall, with few sub-lethal effects. A 0·5 ms duration square pulse waveform had significantly higher catch-per-unit-power than the other waveforms.     

The effect of polymyxin B nonapeptide (PMBN) on transformation

The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E. coli HB101 with pBR322 plasmid DNA was investigated. Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse. In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.     

Electrotransformation of Clostridium thermocellum

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.     

Improved cloning vectors and transformation procedure for Lactococcus lactis

Four shuttle vectors (pMIG 1, 2, 2H and 3) have been constructed based on the broad host-range plasmid pCK1. All the pMIG vectors possess a multiple cloning site containing 12 or more unique restriction enzyme sites, and are stably maintained at either high or low copy number in Lactococcus lactis and in Escherichia coli. By cloning the E. coli pUC replicon into one of these vectors a plasmid was constructed which can replicate to high copy number in recA strains of E. coli. The broad host-range of the pCK1 replicon may enable these cloning vectors to be used in a number of Gram-positive bacteria. One of these vectors was used to optimize an electroporation procedure for transformation of a commonly used plasmid-cured strain MGI363 of L. lactis which routinely yielded 1 times 10⁷ to 5 times 10⁷ transformants μg^-1 supercoiled DNA using stored, snap-frozen cells. This transformation efficiency was obtained by growing the cells in medium containing the cell wall weakening agent glycine, to an upper limit of 2·5% w.v. Although growth of L. lactis strain MGI363 was inhibited by the use of 0·5 mol 1^-1 sucrose as an osmotic stabilizer, the presence of sucrose in the electroporation buffer was critical for high transformation efficiency. Other variables which were tested for their effect on the efficiency of transformation were cell concentration, DNA concentration, pulse time and field strength. These results provide a model procedure which can be followed to optimize conditions for the genetic transformation of various strains of L. lactis.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei.

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

A new plasmid, pA387, has been isolated from "Amycolatopsis sp." (DSM 43387). This plasmid could be isolated from liquid culture as well as mycelium from agar plates by a modified procedure. Plasmid pA387 is about 29.6 kb and can be cured at low frequency by protoplasting and ethidium bromide and heat treatment. Hybridization experiments showed that this plasmid is present in free form and does not integrate into the chromosome. A hybrid plasmid was constructed by cloning a 5.1-kb fragment of pA387 into the Escherichia coli vector pDM10. This hybrid plasmid, termed pRL1, could be transformed into Amycolatopsis mediterranei and A. orientalis by electroporation. A transformation frequency of 2.2 x 10(3) transformants per micrograms of DNA at 12.5 kV/cm and a pulse duration of 10.8 ms was obtained in A. mediterranei, whereas 1.1 x 10(5) transformants per microgram of DNA were obtained at a field strength of 7.5 kV/cm and a pulse duration of 7.6 ms in A. orientalis. Plasmid pRL1 is the first hybrid plasmid which could be used successfully for the transformation of A. mediterranei. The plasmid has a rather high copy number, is genetically stable, and can be easily reisolated from A. mediterranei. Plasmid pRL1 will be useful for further construction of a shuttle vector for E. coli and A. mediterranei and becomes the basis for the development of gene cloning techniques in Amycolatopsis spp.     

The effects of pulsed electromagnetic fields on the cellular activity of SaOS-2 cells

Although pulsed electromagnetic fields (PEMFs) have been used for treatments of nonunion bone fracture healing for more than three decades, the underlying cellular mechanism of bone formation promoted by PEMFs is still unclear. It has been observed that a series of parameters such as pulse shape and frequency should be carefully controlled to achieve effective treatments. In this article, the effects of PEMFs with repetitive pulse burst waveform on the cellular activity of SaOS-2 osteoblast-like cells were investigated. In particular, cell proliferation and mineralization due to the imposed PEMFs were assessed through direct cell counts, the MTT assay, tissue nonspecific alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining. PEMF stimulation with repetitive pulse burst waveform did not affect metabolic activity and cell number. However, the ALP activity of SaOS-2 cells and mineral nodule formation increased significantly after PEMF stimulation. These observations suggest that repetitive pulse burst PEMF does not affect cellular metabolism; however, it may play a role in the enhancement of SaOS-2 cell mineralization. We are currently investigating cellular responses under different PEMF waveforms and Western blots for protein expression of bone mineralization specific proteins.     

Fast kinetics studies of Escherichia coli electrotransformation.

Direct gene transfer is achieved in Escherichia coli by use of square wave electric pulsing. As observed by video monitoring, the field pulse causes bacteria to orientate parallel to the field lines. Rapid kinetic turbidity changes indicate that this process happens quickly. In these circumstances, and in pulsing conditions prone to inducing transformation, only caps are affected by the field. Considerable cytoplasmic ion leakage occurs during the pulse, affecting the interfacial ionic concentration. The pulsing-buffer osmolarity has to be close to that used with protoplasts. Contact between the plasmid and the bacteria can be very short before the pulse but must be present during the pulse. The plasmid remains accessible to externally added DNases up to 5 days after the pulse, suggesting that the transfer step is slow. Electric-field-mediated transfer can be described in two steps: the anchoring process during the pulse, followed by the crossing of the membrane.     

Assessment of pulse rate variability by the method of pulse frequency demodulation

Background  

Due to its easy applicability, pulse wave has been proposed as a surrogate of electrocardiogram (ECG) for the analysis of heart rate variability (HRV). However, its smoother waveform precludes accurate measurement of pulse-to-pulse interval by fiducial-point algorithms. Here we report a pulse frequency demodulation (PFDM) technique as a method for extracting instantaneous pulse rate function directly from pulse wave signal and its usefulness for assessing pulse rate variability (PRV).     

Electrooptics studies of Escherichia coli electropulsation: orientation, permeabilization, and gene transfer.

Fast optical transient signals are suitable approaches to the investigation of the behavior of bacteria during an electric pulse. In a previous work, by a dual approach taking advantage of a video method and a fast kinetic study of the light transmitted across a cell suspension, we showed that a field-induced orientation phenomenon was affecting the rod-shaped bacteria during the pulse (Eynard et al., 1992. Eur. J. Biochem. 209:431-436). In the present work, time courses of electro-induced responses of bacteria during a single square-wave pulse are analyzed. Observations of both the orientation step and the permeabilization process are relevant. These two steps are affected by the addition of DNA. They both obey to a first-order kinetic. The conclusion of this work is that Escherichia coli permeabilization and transformation are multistep processes: orientation (step 1) is followed by an envelope alteration (step 2), all steps being affected by plasmid addition. In the case of E. coli, a rod-shaped bacteria, the orientation process (step 1) brings the cell parallel to the field direction. The pulse duration must be longer than the orientation characteristic time (approximately 1 ms) to trigger an effective permeabilization and its associated events. The permeabilization process (step 2) is associated with a field-induced dipole effect.     

Role of spontaneous current oscillations during high-efficiency electrotransformation of thermophilic anaerobes

Current oscillations at about 24 MHz were observed during electrotransformation (ET) of the thermophilic anaerobes Clostridium thermocellum ATCC 27405, C. thermocellum DSM 1313, and Thermoanaerobacterium saccharolyticum YS 485, using a pulse gated by a square signal generated by a custom generator. In experiments in which only the field strength was varied, all three of these strains resulted in a one-to-one correspondence between the appearance of current oscillations and successful ET. Oscillations accompanied ET of both C. thermocellum strains only at field strengths of > or =12 kV/cm, and ET was only observed above the same threshold. Similarly, for T. saccharolyticum, oscillations were only observed at field strengths of > or =10 kV/cm, and ET was only observed above the same threshold. When a passive electrical filter consisting of an inductor and resistor in parallel was added to the system to prevent the development of oscillations, ET efficiencies were reduced dramatically for all three strains at all field strengths tested. The maximum tested field strength, 25 kV/cm, resulted in the maximum measured transformation efficiency for all three strains. At this field strength, the efficiency of ET in the absence of oscillations was decreased compared to that observed in the presence of oscillations by 500-fold for C. thermocellum ATCC 27405, 2,500-fold for C. thermocellum DSM 1313, and 280-fold for T. saccharolyticum. Controls using the same apparatus with Escherichia coli cells or a resistor with a value representative of the direct current resistance of typical cell samples did not develop oscillations, and ET efficiencies obtained with E. coli were the same with or without the electrical filter included in the pulse generator circuit. The results are interpreted to indicate that spontaneously arising oscillations have a large beneficial effect on transformation efficiency in the system employed here and that the development of oscillations in this system is affected by the cell species present.