- Email: [email protected]
Low cost device for electrotransformation and its application to the highly efficient transformation of Escherichia coli and Enterococcus faecalis
PLASMID
26, 131-135 (1991)
Low Cost Device for Electrotransformation Efficient Transformation of Escherichia SHUHEIFUJIMOTO,
and Its Application to the Highly co/i and Enterococcus hem/is
HAJIMEHASHIMOTO,ANDYASUYOSHI
IKE
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan A simple, low cost device for electrotransformation has been designed and constructed. The cost of the circuit was only %150. Maximum field strength of 12,000 V/cm with an actual time constant up to 11 msec was obtained with a newly designed circuitry and a 0. l-cm electrode gap cuvette. Eschericiu coli strains DH 1, DHSa, and LE392 were transformed at an efficiency of lO’/~g DNA with plasmids pUCl8 and pBR322. E.fiecafis strain OGlX was transformed at an efficiency of 0.9 X IO’/pg DNA without treatment with glycine. o 1991 Academic press, IX
The introduction of DNA into bacteria by transformation is an essentialstep in the construction of recombinant strains. Some bacterial speciesare naturally competent for DNA uptake (Stewart and Carlson, 1986), but natural transformation systems have not been demonstrated in the majority of bacterial species. Without a natural transformation system, alternative methods are used to induce competence. One is a treatment of cells with Ca” or other divalent metal cations as in the casewith Escherichia coli (Mandel and Higa, 1970). Another method, mainly used for transformation of certain streptomycetes (Bibb et al., 1978) and B. subtilis (Chang and Cohen, 1979) is to make protoplasts by removing the bacterial cell wall. DNA then is introduced into the protoplasts by the action of a fusant such aspolyethylene glycol. Methods of such chemical treatments yielding 1OS to lo9 transformants/pg DNA with several strains of E. coli (Hanahan, 1983) and 10’ to lo6 transformants/pg DNA with E. faecalis (Wirth et al., 1986) have been described, but the preparation of the highly competent cells is affected by many factors and it is difficult to obtain such high efficiencies routinely. Recently, electrotransformation has become a well-known method for transformation of eukaryotic (Potter et al., 1984; Fromm et al., 1985) and prokaryotic cells (Miller et al., 1988). This process facilitates 131
entry of DNA into cells during their exposure to electrical fields of very high amplitude. This method yields higher transformation efficiencies than those obtained by chemical treatments. E. coli strain DHSa and LE392 have been transformed at efficiencies of lo9 to lO’O/pg DNA (Dower et al., 1988), and glycine-treated E. faecalis strain JH2-2 has been transformed to the efficiency of 106/pg DNA (Cruz-Rodz and Gilmore, 1990). The method is simple and consistent; however, the hardware is expensive ($1000 - $10000; Chassy et al., 1988). We describe here a simple and low cost device for electrotransformation and its use in the electrotransformation of E. coli and E. faecalis. With the ability to provide comparable electrical conditions to the commercially available instruments, our device should be useful in the electrotransformation of many other bacterial species that have been described as being transformable by this process.
MATERIALS
AND
METHODS
Bacteria and plasmids. We used E. coli strain DHl (Ham&an, 1983), DH5a (Bethesda Research Laboratories), and LE392 (Stratagene), and E. faecalis strain OGI X (Ike et a/., 1983). Plasmids pBR322 (Bolivar et al., 1977) pUC18 (Norrander et al., 1983), 0147-619X/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.
132
FUJIMOTO,
HASHIMOTO,
and pAM40 1 (10.4 kb, cut tet; Wirth and Clewell, 1986) were used as transforming DNA. Media. LB medium (Luria-Bertani medium, 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl per liter) was used for growth of E. coli with or without 1.5% agar. THB (Todd Hewitt broth, Difco Laboratories, Detroit, MI) was used for growth of E. fuecalis with or without 1.5% agar. SOC (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO,, 10 mM MgCl,, 20 mM glucose) was used for expression medium and AB3 (Antibiotic medium 3, Difco) was used for plasmid DNA preparation.
Growth of bacterial cells for transformation. An over-night culture of E. coli was diluted l/125 into 500 ml of LB medium and incubated at 30°C with shaking. Preliminary experiments showed that the culture at 30°C tended to bear more consistent results than the culture at 37°C. Growth of the culture was monitored with a Klett-Summerson calorimeter with No. 54 filter (Klett Manufacturing Co., Long Island City, NY). The cells were grown 5-8 hours to mid-log growth phase (30-40 Klett units) and harvested by chilling the flask 20 min on ice and then centrifuging at 6000g for 10 min at 4°C. The cells were suspended in 500 ml of ice-cold sterile deionized water, centrifuged as above, suspended in 200 ml of ice-cold sterile deionized water, and again centrifuged. They then were washed twice with 40 ml of sterile deionized water containing 10% glycerol. The cell pellet was suspended in 1 ml of sterile deionized water containing 10% glycerol, divided into 20-~1 aliquots and frozen at -80°C. Cells of E. fueculis were prepared identically except that THB was used for the growth instead of LB medium. Preparation ofplasmid DNA. pBR322 and pUC 18 ( 10 rig/ml) were obtained from Nippon Gene Co., ltd. (Toyama, Japan). pAM401 was prepared from E. coli strain DH 1 harboring pAM40 1 by a small-scale alkaline lysate method described previously (Sambrook et al., 1989) suspended in water, and quantified using a minigel method described previously (Sambrook et al., 1989).
AND
IKE
Electronics. Generally, the electroporation of prokaryotic cells requires high field strength (Chassy et al., 1988). High voltages usually make the power supplies complex and costly. We designed and constructed a simple and inexpensive apparatus (i.e., the cost was $150). The essential part of the electric circuit is shown in Fig. 1. The basic design of the circuit is that it very quickly charges the capacitor of 2.2 PF to desired voltage, and the capacitor discharges through a parallel resistor (Rp) and a sample placed between two electrodes. To avoid the difficulties of switching a high voltage, we used a semiconductor switch (thyristor) to switch the large current of low voltage and converted the current to a high voltage with a transformer. Actual waveforms were observed with an oscilloscope (CS-5 130, Kenwood Corp., Japan). Peak voltages and the duration of pulses also were measured with a peak voltage meter and a voltage-time integrator constructed by us. The voltage-time integrator measures the area under the voltage (v-time(t) curve (AUC) and the result is equal to the the product of a time constant and a peak voltage (shown below). V = V,
X
e@
(7 = time constant, V, = peak voltage) 02 AUC = V.dt s0
=
‘x
V, x e-I” . dt s0 = VOX7 An actual time constant was calculated by dividing AUC by an actual peak voltage. Electrodes. As the field strength in Vcm-’ is in inverse proportion to the gap between the electrodes, the voltage requirements placed on power supply design can be reduced if the spacing between electrodes is made smaller. That is, using the 0. l-cm gap electrodes instead of the standard 0.2-cm gap electrodes, the voltage requirement can be reduced to half. For the purpose of simplicity, we used 0.1 -cm electrode gap Potter-style cuvette (Bio-Rad).
133
NEW DEVICE FOR ELECTROPORATION
OUTPUT 0 Rp:l.OkQ-20ki-J FIG. 1. The essential part of the electric circuit for electrotransformation. The large current of low voltage (-300 V) from the capacitor of 400 PF is switched by a thyristor and is converted to a high voltage by a transformer and it very quickly charges the capacitor of 2.2 pF. Then the capacitor of 2.2 PF discharges through a sample and a parallel resistor (Rp) which is in parallel with the sample.
Electrotransformation. DNA (2~1) in water RESULTS AND DISCUSSION was mixed with an 18 ~1 of thawed cells in a Electrical conditions. The main parame1.5ml microtube, and the mixture was left on ice until the electrotransformation. The ters of electrical condition are the waveform, ice-cold mixture was transferred to a chilled the field strength, and the duration of the pulse. As shown in Fig. 2, the device genercuvette and a single electric pulse was ap plied. Immediately following the discharge, 1 ates a pulse of an exponential decay waveml of SOC was added into the cuvette and form. The voltage rises to a peak very quickly mixed using a micropipette. In the experi- (within 500 psec) and declines according to ment with E. coli, the mixture was trans- the time constant. We used a standard type ferred to a 17 X 100 mm polypropylene tube power supply (300 V, 100 mA, for electrophoand incubated for 60 min at 37°C with vigorresis) as an external electric supply for the cirous shaking. In the experiment with E.faeca- cuit. The peak voltage was in proportion to Zis,the mixture was transferred to 1.5-ml mi- the voltage supplied to the circuit. The maxicrotube and incubated for 90 min at 37°C mum peak voltage of 1.2 kV was generated without shaking. After the incubation for expression, cells were diluted appropriately in SOC and 0.1 ml samples of cell suspension were spread on plates containing selective drugs. Transformants of E. coli with pUC 18 and pBR322 were selected on LB medium with agar containing ampicillin ( 100 pg/ml) and tetracycline ( 10 pg/ml). Transformants of E. faecalis containing pAM401 were selected on THB agar containing chloramphenicol (10 pg/ml). The selective plates for E. coli or E.faecalis were incubated for 12 to 16 h or 12 to 48 h, respectively. Controls were included in experimental trials in which either the electric pulse or plasmid DNA was omitted. Transformation was confirmed by FIG. 2. An exponential decay pulse with the time condetecting plasmid DNA in small-scale lysate stant of 11 msec generated from the circuit shown in Fig. oftransformants using agarose gel electropho1. The scale of the oscilloscope was set at 0.2 kV/division and 5 msec/division. resis.
134
PUJIMOTO,
HASHIMOTO, TABLE
AND IKE
1
ELECTROTRANSFORMATION OF E. coli” Strain
Plasmid
DHI
pUC18 pBR322 pUC18 pBR322 pUC18 pBR322
DHSol LE392
Efficiency (/pg DNA) 2.0 x 1.3 x 1.5 x 0.5 x 6.4 x 4.4 x
109 109 lo9 lo9 109 lo9
a Plasmid DNA (20 pg) was added to 20 ~1 of cell suspensions (2.2 - 5.0 x 10” cells/ml) and pulsed at the field strength of 12 kV/cm with actual time constants of 7 - 8 msec with parallel resistor of 4.7 kR. Transformants were selected on ampicillin (pUC 18) or tetracycline (pBR322).
when 300 V was supplied to the circuit. The peak voltage of 1.2 kV corresponded to the field strength of 12 kV/cm using the 0. l-cm electrode gap cuvette. The change of the actual peak voltage caused by the change of other conditions was within 0.05 kV and was thought to be negligible. The duration of the pulse was controlled by changing the resistor which was set in parallel with the sample (parallel resistor). Without a sample, the actual time constants were equal to the time constants calculated as a product of the resistance of the parallel resistor and the capacitance of the capacitor (2.2 PF). With samples, actual time constants were reduced. In the case with E. coli strain DH 1, actual time constants obtained were 4, 7,9, and 11 msec using the parallel resistor of 2.2,4.7, 10, and 20 kQ, respectively. The actual time constants obtained in the three different E. coli strains
of DHl, DHSa, and LE392 were similar (7, 8, and 7 msec, respectively, with the 4.7 kfi parallel resistor). In the case with E. faecalies strain OG 1X, actual time constants obtained were 2,3, and 4 msec using the resistor of 4.7, 10, and 20 kQ, respectively. In all these cases, the static resistance of the sample in the cuvette was more than 500 kC$ so the reduction of the time constant was thought to be the result of the increase of the conductivity of the sample during the electroporation. The exponential decay waveform pulse with a maximum field strength of 12 kV/cm and an actual time constant up to 11 msec was compatible to formerly described or commercially available devices. This means that our hardware can be directly applied to the transformation of many other bacterial species that have been described as transformable by electrotransformation.
TABLE 2 EFFE~XOF THE ACTUAL TIMECONSTANTONELE~ROTRANSFORMATIONOF Parallel resistor (W
Actual time constant (m=c)
2.2 4.7 10 20
4 7 9 11
E. coli” Efficiency C/M DNA) 0.6 x 1.3 x 2.6 x 1.2 x
lo9 lo9 lo9 IO9
’ An actual peak voltage and an area under the voltage-time curve (AUC) were measured. An actual time constant was calculated by dividing AUC by an actual peak voltage. pBR322 DNA (20 pg) was added to 20 ~1 of DH 1 cell suspensions (2.2 X 10” cells/ml) and pulsed at the field strength of 12 kV/cm with the indicated parallel resistor. Transformants were selected on tetracycline.
135
NEW DEVICE FOR ELECTROPORATION
Electrotransformation of E. coli. Three strains of E. coli were transformed at an efficiency of 109/pg DNA using pUCl8 and pBR322 (Table 1). LE392 was transformed at an efficiency of 6.4 X 109/hg DNA with pUC 18. Table 2 shows the effect of the actual time constant on the electrotransformation. Transformation with the actual time constant of 9 msec controlled by the 10 kQ parallel resistor produced the maximum transformation efficiency. Electrotransformation of E. faecalis. pAM401 (28 ng) was added to OGlX cell suspension and pulsed at field strength of 12 kV/cm. The maximum transformation efficiency of 0.9 X lO’/pg DNA was obtained when the actual time constant was 4 msec with 20 kQ parallel resistor. Cruz-Rodz and Gilmore ( 1990) transformed glycine-treated E. faecalis strain JH2-2 at the efficiency of 106/pg DNA. Compared to their method with glycine-treated cells, our method using the cells without glycine treatment is less efficient but much more simple and time saving. We have described new hardware for electrotransformation, its comparability to the formerly used devices, and its application to the highly efficient transformation of E. coli and E. faecalis. The procedure and the device described here are simple and inexpensive. We hope that our system will make electrotransformation a more accessible technology. ACKNOWLEDGMENT This work was supported in part by Grant 03304030 from the Ministry of Education, Science, and Culture of Japan.
REFERENCES BIBB, M. J., WARD, J. M., AND HOPWOOD, D. A. (1978). Transformation of plasmid DNA into Streptomyces at high frequency. Nature 274,398-400. BOLIVAR, F., RODRIGUEZ, R. L., GREEN, P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W.,
CROSA, J. H., AND FALKOW, S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95- 113. CHANG, S., AND COHEN, S. N. ( 1979). High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168. 11 l-l 15. CHASSY, B. M., MERCENIER, A., &D FLICKINGER, J. ( 1988). Transformation of bacteria by electroporation. Trends
Biotechnol.
6, 303-309.
CRUZ-RODS, A. L., AND GILMORE, M. S. (1990). High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol. Gen. Genet. 224, 152-154. DOWER, W. J., MILLER, J. F., AND RAGSDALE, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acid Res. 16, 6127-6145. FROMM, M., TAYLOR, L. P., AND WALBOT, V. (1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82,5824-5828.
HANAHAN, D. ( 1983). Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166,557-580. IKE, Y., CRAIG, R. A., WHITE, B. A., YAGI, Y., AND CLEWELL, D. B. (1983). Modification of Streptococcus faecahs sex pheromones after acquisition of plasmid DNA. Proc. Natl. Acad. Sci. USA 80, 5369-5373. MANDEL, M., AND HIGA, A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159162. MILLER, J. F., DOWER, W. J., AND TOMPKINS, L. S. ( 1988). High-voltage electroporation of bacteria: Genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. USA 85, 856860. NORRANDER, J., KEMPE, T., AND MESSING, J. (1983). Construction of improved Ml3 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 10 l106. POTTER, H., WEIR, L., AND LEDER, P. ( 1984). Enhancerdependent expression of human K immunoglobulin genes introduced into mouSe pro-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81,7 16 l7165. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). “Molecular Cloning. A Laboratory Manual,” (2nd Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. STEWART, G. J., AND CARLSON, C. A. (1986). The biology of natural transformation. Annu. Rev. Microbial. 40,21 l-235. WIRTH, R., AN, F. Y., AND CLEWELL, D. B. (1986). Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecahs shuttle vector. J. Bacterial. 165, 831-836.
26, 131-135 (1991)
Low Cost Device for Electrotransformation Efficient Transformation of Escherichia SHUHEIFUJIMOTO,
and Its Application to the Highly co/i and Enterococcus hem/is
HAJIMEHASHIMOTO,ANDYASUYOSHI
IKE
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan A simple, low cost device for electrotransformation has been designed and constructed. The cost of the circuit was only %150. Maximum field strength of 12,000 V/cm with an actual time constant up to 11 msec was obtained with a newly designed circuitry and a 0. l-cm electrode gap cuvette. Eschericiu coli strains DH 1, DHSa, and LE392 were transformed at an efficiency of lO’/~g DNA with plasmids pUCl8 and pBR322. E.fiecafis strain OGlX was transformed at an efficiency of 0.9 X IO’/pg DNA without treatment with glycine. o 1991 Academic press, IX
The introduction of DNA into bacteria by transformation is an essentialstep in the construction of recombinant strains. Some bacterial speciesare naturally competent for DNA uptake (Stewart and Carlson, 1986), but natural transformation systems have not been demonstrated in the majority of bacterial species. Without a natural transformation system, alternative methods are used to induce competence. One is a treatment of cells with Ca” or other divalent metal cations as in the casewith Escherichia coli (Mandel and Higa, 1970). Another method, mainly used for transformation of certain streptomycetes (Bibb et al., 1978) and B. subtilis (Chang and Cohen, 1979) is to make protoplasts by removing the bacterial cell wall. DNA then is introduced into the protoplasts by the action of a fusant such aspolyethylene glycol. Methods of such chemical treatments yielding 1OS to lo9 transformants/pg DNA with several strains of E. coli (Hanahan, 1983) and 10’ to lo6 transformants/pg DNA with E. faecalis (Wirth et al., 1986) have been described, but the preparation of the highly competent cells is affected by many factors and it is difficult to obtain such high efficiencies routinely. Recently, electrotransformation has become a well-known method for transformation of eukaryotic (Potter et al., 1984; Fromm et al., 1985) and prokaryotic cells (Miller et al., 1988). This process facilitates 131
entry of DNA into cells during their exposure to electrical fields of very high amplitude. This method yields higher transformation efficiencies than those obtained by chemical treatments. E. coli strain DHSa and LE392 have been transformed at efficiencies of lo9 to lO’O/pg DNA (Dower et al., 1988), and glycine-treated E. faecalis strain JH2-2 has been transformed to the efficiency of 106/pg DNA (Cruz-Rodz and Gilmore, 1990). The method is simple and consistent; however, the hardware is expensive ($1000 - $10000; Chassy et al., 1988). We describe here a simple and low cost device for electrotransformation and its use in the electrotransformation of E. coli and E. faecalis. With the ability to provide comparable electrical conditions to the commercially available instruments, our device should be useful in the electrotransformation of many other bacterial species that have been described as being transformable by this process.
MATERIALS
AND
METHODS
Bacteria and plasmids. We used E. coli strain DHl (Ham&an, 1983), DH5a (Bethesda Research Laboratories), and LE392 (Stratagene), and E. faecalis strain OGI X (Ike et a/., 1983). Plasmids pBR322 (Bolivar et al., 1977) pUC18 (Norrander et al., 1983), 0147-619X/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.
132
FUJIMOTO,
HASHIMOTO,
and pAM40 1 (10.4 kb, cut tet; Wirth and Clewell, 1986) were used as transforming DNA. Media. LB medium (Luria-Bertani medium, 10 g Bacto tryptone, 5 g Bacto yeast extract, 10 g NaCl per liter) was used for growth of E. coli with or without 1.5% agar. THB (Todd Hewitt broth, Difco Laboratories, Detroit, MI) was used for growth of E. fuecalis with or without 1.5% agar. SOC (2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgSO,, 10 mM MgCl,, 20 mM glucose) was used for expression medium and AB3 (Antibiotic medium 3, Difco) was used for plasmid DNA preparation.
Growth of bacterial cells for transformation. An over-night culture of E. coli was diluted l/125 into 500 ml of LB medium and incubated at 30°C with shaking. Preliminary experiments showed that the culture at 30°C tended to bear more consistent results than the culture at 37°C. Growth of the culture was monitored with a Klett-Summerson calorimeter with No. 54 filter (Klett Manufacturing Co., Long Island City, NY). The cells were grown 5-8 hours to mid-log growth phase (30-40 Klett units) and harvested by chilling the flask 20 min on ice and then centrifuging at 6000g for 10 min at 4°C. The cells were suspended in 500 ml of ice-cold sterile deionized water, centrifuged as above, suspended in 200 ml of ice-cold sterile deionized water, and again centrifuged. They then were washed twice with 40 ml of sterile deionized water containing 10% glycerol. The cell pellet was suspended in 1 ml of sterile deionized water containing 10% glycerol, divided into 20-~1 aliquots and frozen at -80°C. Cells of E. fueculis were prepared identically except that THB was used for the growth instead of LB medium. Preparation ofplasmid DNA. pBR322 and pUC 18 ( 10 rig/ml) were obtained from Nippon Gene Co., ltd. (Toyama, Japan). pAM401 was prepared from E. coli strain DH 1 harboring pAM40 1 by a small-scale alkaline lysate method described previously (Sambrook et al., 1989) suspended in water, and quantified using a minigel method described previously (Sambrook et al., 1989).
AND
IKE
Electronics. Generally, the electroporation of prokaryotic cells requires high field strength (Chassy et al., 1988). High voltages usually make the power supplies complex and costly. We designed and constructed a simple and inexpensive apparatus (i.e., the cost was $150). The essential part of the electric circuit is shown in Fig. 1. The basic design of the circuit is that it very quickly charges the capacitor of 2.2 PF to desired voltage, and the capacitor discharges through a parallel resistor (Rp) and a sample placed between two electrodes. To avoid the difficulties of switching a high voltage, we used a semiconductor switch (thyristor) to switch the large current of low voltage and converted the current to a high voltage with a transformer. Actual waveforms were observed with an oscilloscope (CS-5 130, Kenwood Corp., Japan). Peak voltages and the duration of pulses also were measured with a peak voltage meter and a voltage-time integrator constructed by us. The voltage-time integrator measures the area under the voltage (v-time(t) curve (AUC) and the result is equal to the the product of a time constant and a peak voltage (shown below). V = V,
X
e@
(7 = time constant, V, = peak voltage) 02 AUC = V.dt s0
=
‘x
V, x e-I” . dt s0 = VOX7 An actual time constant was calculated by dividing AUC by an actual peak voltage. Electrodes. As the field strength in Vcm-’ is in inverse proportion to the gap between the electrodes, the voltage requirements placed on power supply design can be reduced if the spacing between electrodes is made smaller. That is, using the 0. l-cm gap electrodes instead of the standard 0.2-cm gap electrodes, the voltage requirement can be reduced to half. For the purpose of simplicity, we used 0.1 -cm electrode gap Potter-style cuvette (Bio-Rad).
133
NEW DEVICE FOR ELECTROPORATION
OUTPUT 0 Rp:l.OkQ-20ki-J FIG. 1. The essential part of the electric circuit for electrotransformation. The large current of low voltage (-300 V) from the capacitor of 400 PF is switched by a thyristor and is converted to a high voltage by a transformer and it very quickly charges the capacitor of 2.2 pF. Then the capacitor of 2.2 PF discharges through a sample and a parallel resistor (Rp) which is in parallel with the sample.
Electrotransformation. DNA (2~1) in water RESULTS AND DISCUSSION was mixed with an 18 ~1 of thawed cells in a Electrical conditions. The main parame1.5ml microtube, and the mixture was left on ice until the electrotransformation. The ters of electrical condition are the waveform, ice-cold mixture was transferred to a chilled the field strength, and the duration of the pulse. As shown in Fig. 2, the device genercuvette and a single electric pulse was ap plied. Immediately following the discharge, 1 ates a pulse of an exponential decay waveml of SOC was added into the cuvette and form. The voltage rises to a peak very quickly mixed using a micropipette. In the experi- (within 500 psec) and declines according to ment with E. coli, the mixture was trans- the time constant. We used a standard type ferred to a 17 X 100 mm polypropylene tube power supply (300 V, 100 mA, for electrophoand incubated for 60 min at 37°C with vigorresis) as an external electric supply for the cirous shaking. In the experiment with E.faeca- cuit. The peak voltage was in proportion to Zis,the mixture was transferred to 1.5-ml mi- the voltage supplied to the circuit. The maxicrotube and incubated for 90 min at 37°C mum peak voltage of 1.2 kV was generated without shaking. After the incubation for expression, cells were diluted appropriately in SOC and 0.1 ml samples of cell suspension were spread on plates containing selective drugs. Transformants of E. coli with pUC 18 and pBR322 were selected on LB medium with agar containing ampicillin ( 100 pg/ml) and tetracycline ( 10 pg/ml). Transformants of E. faecalis containing pAM401 were selected on THB agar containing chloramphenicol (10 pg/ml). The selective plates for E. coli or E.faecalis were incubated for 12 to 16 h or 12 to 48 h, respectively. Controls were included in experimental trials in which either the electric pulse or plasmid DNA was omitted. Transformation was confirmed by FIG. 2. An exponential decay pulse with the time condetecting plasmid DNA in small-scale lysate stant of 11 msec generated from the circuit shown in Fig. oftransformants using agarose gel electropho1. The scale of the oscilloscope was set at 0.2 kV/division and 5 msec/division. resis.
134
PUJIMOTO,
HASHIMOTO, TABLE
AND IKE
1
ELECTROTRANSFORMATION OF E. coli” Strain
Plasmid
DHI
pUC18 pBR322 pUC18 pBR322 pUC18 pBR322
DHSol LE392
Efficiency (/pg DNA) 2.0 x 1.3 x 1.5 x 0.5 x 6.4 x 4.4 x
109 109 lo9 lo9 109 lo9
a Plasmid DNA (20 pg) was added to 20 ~1 of cell suspensions (2.2 - 5.0 x 10” cells/ml) and pulsed at the field strength of 12 kV/cm with actual time constants of 7 - 8 msec with parallel resistor of 4.7 kR. Transformants were selected on ampicillin (pUC 18) or tetracycline (pBR322).
when 300 V was supplied to the circuit. The peak voltage of 1.2 kV corresponded to the field strength of 12 kV/cm using the 0. l-cm electrode gap cuvette. The change of the actual peak voltage caused by the change of other conditions was within 0.05 kV and was thought to be negligible. The duration of the pulse was controlled by changing the resistor which was set in parallel with the sample (parallel resistor). Without a sample, the actual time constants were equal to the time constants calculated as a product of the resistance of the parallel resistor and the capacitance of the capacitor (2.2 PF). With samples, actual time constants were reduced. In the case with E. coli strain DH 1, actual time constants obtained were 4, 7,9, and 11 msec using the parallel resistor of 2.2,4.7, 10, and 20 kQ, respectively. The actual time constants obtained in the three different E. coli strains
of DHl, DHSa, and LE392 were similar (7, 8, and 7 msec, respectively, with the 4.7 kfi parallel resistor). In the case with E. faecalies strain OG 1X, actual time constants obtained were 2,3, and 4 msec using the resistor of 4.7, 10, and 20 kQ, respectively. In all these cases, the static resistance of the sample in the cuvette was more than 500 kC$ so the reduction of the time constant was thought to be the result of the increase of the conductivity of the sample during the electroporation. The exponential decay waveform pulse with a maximum field strength of 12 kV/cm and an actual time constant up to 11 msec was compatible to formerly described or commercially available devices. This means that our hardware can be directly applied to the transformation of many other bacterial species that have been described as transformable by electrotransformation.
TABLE 2 EFFE~XOF THE ACTUAL TIMECONSTANTONELE~ROTRANSFORMATIONOF Parallel resistor (W
Actual time constant (m=c)
2.2 4.7 10 20
4 7 9 11
E. coli” Efficiency C/M DNA) 0.6 x 1.3 x 2.6 x 1.2 x
lo9 lo9 lo9 IO9
’ An actual peak voltage and an area under the voltage-time curve (AUC) were measured. An actual time constant was calculated by dividing AUC by an actual peak voltage. pBR322 DNA (20 pg) was added to 20 ~1 of DH 1 cell suspensions (2.2 X 10” cells/ml) and pulsed at the field strength of 12 kV/cm with the indicated parallel resistor. Transformants were selected on tetracycline.
135
NEW DEVICE FOR ELECTROPORATION
Electrotransformation of E. coli. Three strains of E. coli were transformed at an efficiency of 109/pg DNA using pUCl8 and pBR322 (Table 1). LE392 was transformed at an efficiency of 6.4 X 109/hg DNA with pUC 18. Table 2 shows the effect of the actual time constant on the electrotransformation. Transformation with the actual time constant of 9 msec controlled by the 10 kQ parallel resistor produced the maximum transformation efficiency. Electrotransformation of E. faecalis. pAM401 (28 ng) was added to OGlX cell suspension and pulsed at field strength of 12 kV/cm. The maximum transformation efficiency of 0.9 X lO’/pg DNA was obtained when the actual time constant was 4 msec with 20 kQ parallel resistor. Cruz-Rodz and Gilmore ( 1990) transformed glycine-treated E. faecalis strain JH2-2 at the efficiency of 106/pg DNA. Compared to their method with glycine-treated cells, our method using the cells without glycine treatment is less efficient but much more simple and time saving. We have described new hardware for electrotransformation, its comparability to the formerly used devices, and its application to the highly efficient transformation of E. coli and E. faecalis. The procedure and the device described here are simple and inexpensive. We hope that our system will make electrotransformation a more accessible technology. ACKNOWLEDGMENT This work was supported in part by Grant 03304030 from the Ministry of Education, Science, and Culture of Japan.
REFERENCES BIBB, M. J., WARD, J. M., AND HOPWOOD, D. A. (1978). Transformation of plasmid DNA into Streptomyces at high frequency. Nature 274,398-400. BOLIVAR, F., RODRIGUEZ, R. L., GREEN, P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W.,
CROSA, J. H., AND FALKOW, S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95- 113. CHANG, S., AND COHEN, S. N. ( 1979). High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA. Mol. Gen. Genet. 168. 11 l-l 15. CHASSY, B. M., MERCENIER, A., &D FLICKINGER, J. ( 1988). Transformation of bacteria by electroporation. Trends
Biotechnol.
6, 303-309.
CRUZ-RODS, A. L., AND GILMORE, M. S. (1990). High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol. Gen. Genet. 224, 152-154. DOWER, W. J., MILLER, J. F., AND RAGSDALE, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acid Res. 16, 6127-6145. FROMM, M., TAYLOR, L. P., AND WALBOT, V. (1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82,5824-5828.
HANAHAN, D. ( 1983). Studies on transformation ofEscherichia coli with plasmids. J. Mol. Biol. 166,557-580. IKE, Y., CRAIG, R. A., WHITE, B. A., YAGI, Y., AND CLEWELL, D. B. (1983). Modification of Streptococcus faecahs sex pheromones after acquisition of plasmid DNA. Proc. Natl. Acad. Sci. USA 80, 5369-5373. MANDEL, M., AND HIGA, A. (1970). Calcium-dependent bacteriophage DNA infection. J. Mol. Biol. 53, 159162. MILLER, J. F., DOWER, W. J., AND TOMPKINS, L. S. ( 1988). High-voltage electroporation of bacteria: Genetic transformation of Campylobacter jejuni with plasmid DNA. Proc. Natl. Acad. Sci. USA 85, 856860. NORRANDER, J., KEMPE, T., AND MESSING, J. (1983). Construction of improved Ml3 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 10 l106. POTTER, H., WEIR, L., AND LEDER, P. ( 1984). Enhancerdependent expression of human K immunoglobulin genes introduced into mouSe pro-B lymphocytes by electroporation. Proc. Natl. Acad. Sci. USA 81,7 16 l7165. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). “Molecular Cloning. A Laboratory Manual,” (2nd Ed.). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. STEWART, G. J., AND CARLSON, C. A. (1986). The biology of natural transformation. Annu. Rev. Microbial. 40,21 l-235. WIRTH, R., AN, F. Y., AND CLEWELL, D. B. (1986). Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecahs shuttle vector. J. Bacterial. 165, 831-836.