MicrobiologyLetters 67 (1990) 135-138 Published by Elsevier
135
FEMS
FEMSLE 03870
Genetic transformation of intact ceils of Bacillus subtilis by eleetroporation P. Brigidi 1 E. D e Rossi 2, M.L. Bertarini t, G . Riccardi 2 a n d D . Matteuzzi l J Fe~nentation Chemistry and Industrial Microbiology, Department OfPharmaceutical Sciences, Uniuersity of Bologna, Bologna. and 2 Department of Genetics and Microbiology "A. BIJzzati Traverso; University of Pavia. Pavia, Italy
Received20 July 1989 Revisionreceived20 September 1989 Accepted 27 September 1989 Key words: Bacillus subtilis; Transformation; Electroporation
1. S U M M A R Y Plasmid D N A s were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 104 transformants/~tg DNA. Parameters including growth phase of eafis, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.
2. I N T R O D U C T I O N Bacillus subtilis is a potential model system for biochemical genetics and genetic engineering of Gram-positive bacteria. Plasmid transformation of B. subtilis can be accomplished either with competent cells or by protoplasts in the presence of polyethylene glycol [1].
Correspondence to: D. Matteuzzi, Fermentation Chemistry and
Industrial Microbiology,Department of PharmaceuticalSciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
The elcctroporation technique has recently been introduced to efficiently transform animal, plant and yeast ceils in a simple manner. In addition to eucaryotic ceils, electroporntion has been used to enhance transformation in bacterial systems [2]. As regards the Bacillus genus, the first electropotation report concerned B. cereus protoplasts [3]; more recently B. thuringiensis and 11. cereus intact cells were transformed by electroporation with high efficiencies [4,5]. In this report we describe the use of alectroporation as a rapid, simple and efficient method for transferring genetic information, compared to the transformation systems presently available in B. subtilis. We systematically investigated the effect of several factors on transformation efficiency and up to 10 4 transformants per /tg of plasmid D N A has been routinely obtained.
3, MATERIALS A N D M E T H O D S 3.1. Strain, plasmids and media R subzilis PB14124 (hisH2, metD4, trpC2) was
obtained from our collection and routinely cultured in LI~ medium at 3 0 ° C with shaking. The
0378-1097/90/$03,50 © 1990 Federation of European Microbiological Societies
136 Table 1 Phenotypesand sourcesof plasmid DNAs Plasmid kb Phenotyp¢a/construction Reference pC194 2.9 Cmg [8I pVEI8 4.6 Crag/pC194+ pBCI this study pHVI4 %3 AmpR. CmR/pC194+pBR322 [9l pDM41 9.0 Cmg/pJHl01 + pBCI [7] a Crag: ohlorampheni¢olresistartce; Ampg: ampicillin resistance. plasmids used are listed in Table 1. Plasmid DNA for electroporation was isolated from Escherichia coli or B. subtilis by alkaline lysis followed by purification in a cesium chloride gradient [6]. Plasmid mihiprep DNA from transformants took p l a ~ as described by Maniatis et al. [6]. Restriction enzymes were obtained from Bethesda Research Laboratories, Inc., and were used in the recommended buffers. 3.2. Electroporation Electroporation was carried out using a Bio-Rad Gene PulserS; in some experiments the Pulse Controller was also used. An overnight culture of B. subtilis PB1424 was inoculated in LB broth and grown to an OD6oo of about 0.6, corresponding to mid-log phase. Ceils were chilled on ice for 30 min, harvested by centrifugation, rinsed once in 1 m M Hepes (N-2-hydroxyethylpipesazine-N'-ethanesulfonic acid) pH 7, twice in cold electroporation buffer and resuspended at about 1/30th or 1/200th of the original culture volume, depending on whether or not the pulse controller was used. Electroporation was performed with either fresh cells or with ceils twice frozen and thawed. Three electroporation buffers were used in this study: (I) PEB (272 raM sucrose, ! m M MgCI:, 7 mM potassium phosphate, pH 7.4); (11) HEB (272 mM sucrose, I t a m MgCI2, 7 taM Hepes, pH 7A), and 011) H G (1 mM Hepes, pH 7, 10~ glycerol). For some experiments. PEB and HEB were used at greater than I x buffer/ionic strength (x-fold increased for all buffer components). For electropotation without the pulse controller, plasmid D N A (0.1-10 ~tg in a volume of less than 10 M) was added to 0.8 ml of cells suspended in PEB or HEB (about 9 x 10a-4 x 109 CFU); this mixture
was placed in cold electroporation 0.4 cm cuvette and held on ice for 10 min. Following the application of a high-voltage dectric pulse (25 #F capacitance, 1.2-2.5 kV set voltage), the DNA-cell mixture was incubated on ice for an additional 10 rain, added to 6 ml LB and incubated at 3 0 ° C for 1 h to allow expression of antibiotic resistance prior to plating on LB agar plus 5 /tg/mi of chloramphenicol. Using the pulse controller, 5 0 / t l of cells suspended in H G (2-5 x 10 l° CFU) were mixed with 1 /xl of DNA (100-300 ng), chilled on ice for 1 rain, transferred to a cold 0.2 cm cuvette and subjected to a high-voltage electric pulse (25 pF capacitance, 1.5-2.5 kV set voltage, 100-800 12 resistance). Immediately after the pulse, 1 ml of LB was added and the cell suspension transferred to a sterile tube for incubation at 3 0 ° C for 1 h before plating onto selective media. The ~version frequency (spontaneous mutants) was determined by plating 0.1 ml aliquots of the cell control (no DNA) onto selective media. Control dilutions were also plated on LB agar without antibiotic to determine the number of cells which survive the field pulse treatment. The same procedure was performed with an impulsed control sample. Transformant colonies were visible after 24 h of incubation at 30 ° C.
4. RESULTS A N D DISCUSSION 4.1. Construction of a cloning vector The hybrid plastaid pVEI8 (4.6 kb) was constructed by inserting the small cryptic plasmid pBCI (1.7 kb) [7], linearized with Hhal, into the unique Hhal site of the plasmid pC194 (2.9 kb) [8]. pC194 also replicates in B. subtilis but its replication origin was destroyed by digestion with Hhal. In this way pBCI supplied the Bacillus origin of replication. 4.2. Effect of cell viability, growth phase and concentration Cell viability was not significantly affected by increased electric field strength; in the voltage range tested (3000-12500/cm) the degree of cell death is around 75-85%. The same lethality re-
137 Table 2 Effect of applied voltage on transfo~ation eff~icncy. Cells were petaled in PEB with 0.5 ag of pVEIg DNA Voltage (V/cm)
it/'\ '//\
\
\
3000
Time constant Imsl 7.0
4000
6.7
5000 5000 6250 6250
6.3 6.3 5.4 5,4
Transformation efficiency * 3.9× tO2a 3.7 X 103 a 1.7Xl04a 4.2x103 2,8 x tO4 6.0X tO3
a Using frozen cells. • Efficiencies are expr~.scd ~ number of transformants/pg DNA.
BUFFF.R STRENGTH (X~-OkO)
Fig. l. Transformation efficiency as a function of buffet strenglh. Electroporation conditions: voltage: 6250 V/cm: capacitance: 25 pF: plasmid: pVEI8 (0.5 ~g); buffers: PEB (z~), HEB (0).
suits were o b t a i n e d employing all the various elect r o p o r a t i o n buffers. A m a r k e d increase in transformation efficiency (number of transform a n t s per /tg of plasmid D N A ) was noted with mid-log phase cells at high cell concentrations ( 1 0 9 - 1 0 ~° C F U ) ; as the culture begins to app r o a c h its stationary phase, transformation efficiency decreases.
4. 4. Effect of pulse amplitude and duration As is expected, the voltage used for d e e t r o p o r a lion is the most significant parameter. Initial experiments were carried out without using the pulse controller. T h e effect o f applied v o h a g e o n transformation efficiencies of B. subtilis PB1424 with plasmid p V E I 8 , is reported in T a b l e 2. H i g h e r transformation efficiencies (2.8 × 104) were obtained using the m a x i m u m ek~ctric pulse of 6250 V / c m (time constant about 5 ms) at 2 5 / t F . F u r thermore, we used the Pulse C o n t r o l l e r unit to evaluate the effects of higher voltages, to reduce the risk of arching as well as to allow control of the pulse length. T h e transformation efficiencies obtained are shown in T a b l e 3. O u r results re-
4.3. Effect of buffer composition and concentration As the resistance of the electroporation buffer influences the pulse time constant, two media were tested in experiments without pulse controller. At voltage set between 2.5 and 1.2 kV, lower-resistance buffer P E B generated time constants ranging from 5 . 4 - 7 . 0 ms, while the higherresistance buffet' H E B produced time constants r a n g i n g from 1 0 - 1 6 . 5 ms. T h e effect of varying the buf/er strength on transformation efficiency is shown in Fig. 1. Both H E B and PEB yielded greater n u m b e r o f transformants at 1 x buffer strengths. However, the most conductive buffer P E B in contrast to H E B , was found to increase the transformation efficiency by 10-fold (1.6 x 104 transformants//Lg pVE18).
Table 3 Effect of applied voltage on transformalion efficiencies using Pulse Controller Voltage (V/cm)
Restslance (.q)
Time constant (ms)
Transfermarion efficiency
7500
200 400 600 800 2OO 400 600
4.9 9.1 13.4 17.O 4.9 9.3 13.2
8.5 × 102 3.3x102 1.3X tO2 0 4.3 X 102
200
4.g
0
400
9.3
O
t0ooo
12500
1.3xlO ~
0
Frozen cells were poraled with 0.5 ILg of pVEI8 DNA
138 vealed that no transformants could be selected using electric pulses higher than 10000 V / c m . Also the pulse length had the same dramatic effect on the electroporation efficiency. At 1.5 kV (7 500 V/era), shifting resistance from 200 to 800 fi'+time constant ranged from 4.9 to 17.0 ms; only at this latter value transformation did not occur. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells prior to cleetroporation, repeated freezing and thawing apparently changes the cell wall to a certain extent and facilitates D N A uptake. Furthermore, multiple pulses yielded fewer transformants than single pulses of similar strength (data not shown). Finally, the transformant number is higher when shocked cells are diluted in ratios of 1 : 6 , 1 : 1 0 in LB both prior to incubation as compared with cells diluted in 1 : 1 or 1 : 3 ratios.
4.5. DNA concentration and plasmid size The effect of plasmid D N A concentration on the electroporation efficiency was examined. The number of transformants increases progressively with the addition of between 0.1 and 10 lag of plasmid DNA, but the highest transformation efficiency is obtained with 0.5 lag of added DNA. Four plasmids, i.e. pC194 and pVE18 (from B. subtilis), p H V I 4 and pDM41 (from both E. coil and B. subtilis), were used to determine the effect of plasmid size on the elcctroporation efficiency (Table 4). As expected, smaller plasmid (e.g. pVE18; 4.6 kb) transformed at higher efficiencies than larger plasmid (e.g. pDM41; 9.0 kb).
4.6. Confirmation of plasmid transfer by electropotation Representative transformant colonies were subcultured and subjected to small-vohime lysis to confirm the presence of the appropriate plasmid. The lack of any detectable change in size or endonuclease cutting patterns in the transformed plasmids, demonstrated that deletions or rearrangements did not occur as a consequence of aleetroporation.
4. 7. Conclusion Our report describes the effectiveness of ele~'oporation in B, subtilis PB1424 strain, As
Table 4 Effect of plasmid size on the efficiencyof transformation Plasmid (kb) pC194 (2.9) pVEI8 (4.6) pHVI4 (7.3) pDM41 (9.0)
Transformants/ /~g DNA 9.0 X 103 2.8 X104 1.3× 104 5.0 X l 0 2
Electroporation conditions: voltage:6250 V/era: plasmid concentration: 0.5 ~tg.
regards the Bacillus genus, only B. thuringiensis and B. cereus intact cells had been successfully electroporated [4,5]. In optimizing the method for B. subtilis PB1424, we determined the effect of several variables on transformation efficiency such as growth phase and cell concentration, ionic su~ngth of electroporation buffer, pulse amplitude and duration, D N A concentration and size. The best electroporation efficiency (2.8 x 104) is obtained using exponentially growing cells, frozen and thawed, in PEB buffer, electroporated at 25 laF and 6250 V / c m , with 0.5 lag of the small plasmid pVEI8 (4.6 kb).
REFERENCES It] Bennett, P.M. and Grinsted, J. (1984) Methods in Microbio|ogy, Vol. 17, AcademicPress, London. [2] Luchansky, J.B,, Muriana, P.M. and Klaenhammer. T.R. (1988) Mol. Mierobiol. 2. 637-646. [3] Shivarova,N.. Former, W., Jacob, H.E. and Grigorova, R. (1983) Z. AUg.Mikrobiol. 23. 595-599. [4] Bone, E.J, and Eliot, DJ. 0989) FEMS Microbiol. Leo. S8. 171-178. lSl Belliveao, B.H, and Trevors, J.T. (1989) Appl. Environ. Microbiol. 55,1649-1652. 161 Maniatis, T., Frltseh, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [7] De Rossi, E., Bri$idi. P., Riccardi. G, and Maneuzzl. D. (1989) Corr. Mierobiol. 19.13+19, [81 HorinouchL S. and Weisblum, B. (1982) J. Bacteriol. 150, 815-825. 19] Ehrlich, S.D. (1978) Ptoc. Natl. Acad. Sci. U.S.A. 75, 1433-1436,
135
FEMS
FEMSLE 03870
Genetic transformation of intact ceils of Bacillus subtilis by eleetroporation P. Brigidi 1 E. D e Rossi 2, M.L. Bertarini t, G . Riccardi 2 a n d D . Matteuzzi l J Fe~nentation Chemistry and Industrial Microbiology, Department OfPharmaceutical Sciences, Uniuersity of Bologna, Bologna. and 2 Department of Genetics and Microbiology "A. BIJzzati Traverso; University of Pavia. Pavia, Italy
Received20 July 1989 Revisionreceived20 September 1989 Accepted 27 September 1989 Key words: Bacillus subtilis; Transformation; Electroporation
1. S U M M A R Y Plasmid D N A s were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 104 transformants/~tg DNA. Parameters including growth phase of eafis, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.
2. I N T R O D U C T I O N Bacillus subtilis is a potential model system for biochemical genetics and genetic engineering of Gram-positive bacteria. Plasmid transformation of B. subtilis can be accomplished either with competent cells or by protoplasts in the presence of polyethylene glycol [1].
Correspondence to: D. Matteuzzi, Fermentation Chemistry and
Industrial Microbiology,Department of PharmaceuticalSciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
The elcctroporation technique has recently been introduced to efficiently transform animal, plant and yeast ceils in a simple manner. In addition to eucaryotic ceils, electroporntion has been used to enhance transformation in bacterial systems [2]. As regards the Bacillus genus, the first electropotation report concerned B. cereus protoplasts [3]; more recently B. thuringiensis and 11. cereus intact cells were transformed by electroporation with high efficiencies [4,5]. In this report we describe the use of alectroporation as a rapid, simple and efficient method for transferring genetic information, compared to the transformation systems presently available in B. subtilis. We systematically investigated the effect of several factors on transformation efficiency and up to 10 4 transformants per /tg of plasmid D N A has been routinely obtained.
3, MATERIALS A N D M E T H O D S 3.1. Strain, plasmids and media R subzilis PB14124 (hisH2, metD4, trpC2) was
obtained from our collection and routinely cultured in LI~ medium at 3 0 ° C with shaking. The
0378-1097/90/$03,50 © 1990 Federation of European Microbiological Societies
136 Table 1 Phenotypesand sourcesof plasmid DNAs Plasmid kb Phenotyp¢a/construction Reference pC194 2.9 Cmg [8I pVEI8 4.6 Crag/pC194+ pBCI this study pHVI4 %3 AmpR. CmR/pC194+pBR322 [9l pDM41 9.0 Cmg/pJHl01 + pBCI [7] a Crag: ohlorampheni¢olresistartce; Ampg: ampicillin resistance. plasmids used are listed in Table 1. Plasmid DNA for electroporation was isolated from Escherichia coli or B. subtilis by alkaline lysis followed by purification in a cesium chloride gradient [6]. Plasmid mihiprep DNA from transformants took p l a ~ as described by Maniatis et al. [6]. Restriction enzymes were obtained from Bethesda Research Laboratories, Inc., and were used in the recommended buffers. 3.2. Electroporation Electroporation was carried out using a Bio-Rad Gene PulserS; in some experiments the Pulse Controller was also used. An overnight culture of B. subtilis PB1424 was inoculated in LB broth and grown to an OD6oo of about 0.6, corresponding to mid-log phase. Ceils were chilled on ice for 30 min, harvested by centrifugation, rinsed once in 1 m M Hepes (N-2-hydroxyethylpipesazine-N'-ethanesulfonic acid) pH 7, twice in cold electroporation buffer and resuspended at about 1/30th or 1/200th of the original culture volume, depending on whether or not the pulse controller was used. Electroporation was performed with either fresh cells or with ceils twice frozen and thawed. Three electroporation buffers were used in this study: (I) PEB (272 raM sucrose, ! m M MgCI:, 7 mM potassium phosphate, pH 7.4); (11) HEB (272 mM sucrose, I t a m MgCI2, 7 taM Hepes, pH 7A), and 011) H G (1 mM Hepes, pH 7, 10~ glycerol). For some experiments. PEB and HEB were used at greater than I x buffer/ionic strength (x-fold increased for all buffer components). For electropotation without the pulse controller, plasmid D N A (0.1-10 ~tg in a volume of less than 10 M) was added to 0.8 ml of cells suspended in PEB or HEB (about 9 x 10a-4 x 109 CFU); this mixture
was placed in cold electroporation 0.4 cm cuvette and held on ice for 10 min. Following the application of a high-voltage dectric pulse (25 #F capacitance, 1.2-2.5 kV set voltage), the DNA-cell mixture was incubated on ice for an additional 10 rain, added to 6 ml LB and incubated at 3 0 ° C for 1 h to allow expression of antibiotic resistance prior to plating on LB agar plus 5 /tg/mi of chloramphenicol. Using the pulse controller, 5 0 / t l of cells suspended in H G (2-5 x 10 l° CFU) were mixed with 1 /xl of DNA (100-300 ng), chilled on ice for 1 rain, transferred to a cold 0.2 cm cuvette and subjected to a high-voltage electric pulse (25 pF capacitance, 1.5-2.5 kV set voltage, 100-800 12 resistance). Immediately after the pulse, 1 ml of LB was added and the cell suspension transferred to a sterile tube for incubation at 3 0 ° C for 1 h before plating onto selective media. The ~version frequency (spontaneous mutants) was determined by plating 0.1 ml aliquots of the cell control (no DNA) onto selective media. Control dilutions were also plated on LB agar without antibiotic to determine the number of cells which survive the field pulse treatment. The same procedure was performed with an impulsed control sample. Transformant colonies were visible after 24 h of incubation at 30 ° C.
4. RESULTS A N D DISCUSSION 4.1. Construction of a cloning vector The hybrid plastaid pVEI8 (4.6 kb) was constructed by inserting the small cryptic plasmid pBCI (1.7 kb) [7], linearized with Hhal, into the unique Hhal site of the plasmid pC194 (2.9 kb) [8]. pC194 also replicates in B. subtilis but its replication origin was destroyed by digestion with Hhal. In this way pBCI supplied the Bacillus origin of replication. 4.2. Effect of cell viability, growth phase and concentration Cell viability was not significantly affected by increased electric field strength; in the voltage range tested (3000-12500/cm) the degree of cell death is around 75-85%. The same lethality re-
137 Table 2 Effect of applied voltage on transfo~ation eff~icncy. Cells were petaled in PEB with 0.5 ag of pVEIg DNA Voltage (V/cm)
it/'\ '//\
\
\
3000
Time constant Imsl 7.0
4000
6.7
5000 5000 6250 6250
6.3 6.3 5.4 5,4
Transformation efficiency * 3.9× tO2a 3.7 X 103 a 1.7Xl04a 4.2x103 2,8 x tO4 6.0X tO3
a Using frozen cells. • Efficiencies are expr~.scd ~ number of transformants/pg DNA.
BUFFF.R STRENGTH (X~-OkO)
Fig. l. Transformation efficiency as a function of buffet strenglh. Electroporation conditions: voltage: 6250 V/cm: capacitance: 25 pF: plasmid: pVEI8 (0.5 ~g); buffers: PEB (z~), HEB (0).
suits were o b t a i n e d employing all the various elect r o p o r a t i o n buffers. A m a r k e d increase in transformation efficiency (number of transform a n t s per /tg of plasmid D N A ) was noted with mid-log phase cells at high cell concentrations ( 1 0 9 - 1 0 ~° C F U ) ; as the culture begins to app r o a c h its stationary phase, transformation efficiency decreases.
4. 4. Effect of pulse amplitude and duration As is expected, the voltage used for d e e t r o p o r a lion is the most significant parameter. Initial experiments were carried out without using the pulse controller. T h e effect o f applied v o h a g e o n transformation efficiencies of B. subtilis PB1424 with plasmid p V E I 8 , is reported in T a b l e 2. H i g h e r transformation efficiencies (2.8 × 104) were obtained using the m a x i m u m ek~ctric pulse of 6250 V / c m (time constant about 5 ms) at 2 5 / t F . F u r thermore, we used the Pulse C o n t r o l l e r unit to evaluate the effects of higher voltages, to reduce the risk of arching as well as to allow control of the pulse length. T h e transformation efficiencies obtained are shown in T a b l e 3. O u r results re-
4.3. Effect of buffer composition and concentration As the resistance of the electroporation buffer influences the pulse time constant, two media were tested in experiments without pulse controller. At voltage set between 2.5 and 1.2 kV, lower-resistance buffer P E B generated time constants ranging from 5 . 4 - 7 . 0 ms, while the higherresistance buffet' H E B produced time constants r a n g i n g from 1 0 - 1 6 . 5 ms. T h e effect of varying the buf/er strength on transformation efficiency is shown in Fig. 1. Both H E B and PEB yielded greater n u m b e r o f transformants at 1 x buffer strengths. However, the most conductive buffer P E B in contrast to H E B , was found to increase the transformation efficiency by 10-fold (1.6 x 104 transformants//Lg pVE18).
Table 3 Effect of applied voltage on transformalion efficiencies using Pulse Controller Voltage (V/cm)
Restslance (.q)
Time constant (ms)
Transfermarion efficiency
7500
200 400 600 800 2OO 400 600
4.9 9.1 13.4 17.O 4.9 9.3 13.2
8.5 × 102 3.3x102 1.3X tO2 0 4.3 X 102
200
4.g
0
400
9.3
O
t0ooo
12500
1.3xlO ~
0
Frozen cells were poraled with 0.5 ILg of pVEI8 DNA
138 vealed that no transformants could be selected using electric pulses higher than 10000 V / c m . Also the pulse length had the same dramatic effect on the electroporation efficiency. At 1.5 kV (7 500 V/era), shifting resistance from 200 to 800 fi'+time constant ranged from 4.9 to 17.0 ms; only at this latter value transformation did not occur. Transformation efficiency can be increased by an additional freeze and thaw cycle of the cells prior to cleetroporation, repeated freezing and thawing apparently changes the cell wall to a certain extent and facilitates D N A uptake. Furthermore, multiple pulses yielded fewer transformants than single pulses of similar strength (data not shown). Finally, the transformant number is higher when shocked cells are diluted in ratios of 1 : 6 , 1 : 1 0 in LB both prior to incubation as compared with cells diluted in 1 : 1 or 1 : 3 ratios.
4.5. DNA concentration and plasmid size The effect of plasmid D N A concentration on the electroporation efficiency was examined. The number of transformants increases progressively with the addition of between 0.1 and 10 lag of plasmid DNA, but the highest transformation efficiency is obtained with 0.5 lag of added DNA. Four plasmids, i.e. pC194 and pVE18 (from B. subtilis), p H V I 4 and pDM41 (from both E. coil and B. subtilis), were used to determine the effect of plasmid size on the elcctroporation efficiency (Table 4). As expected, smaller plasmid (e.g. pVE18; 4.6 kb) transformed at higher efficiencies than larger plasmid (e.g. pDM41; 9.0 kb).
4.6. Confirmation of plasmid transfer by electropotation Representative transformant colonies were subcultured and subjected to small-vohime lysis to confirm the presence of the appropriate plasmid. The lack of any detectable change in size or endonuclease cutting patterns in the transformed plasmids, demonstrated that deletions or rearrangements did not occur as a consequence of aleetroporation.
4. 7. Conclusion Our report describes the effectiveness of ele~'oporation in B, subtilis PB1424 strain, As
Table 4 Effect of plasmid size on the efficiencyof transformation Plasmid (kb) pC194 (2.9) pVEI8 (4.6) pHVI4 (7.3) pDM41 (9.0)
Transformants/ /~g DNA 9.0 X 103 2.8 X104 1.3× 104 5.0 X l 0 2
Electroporation conditions: voltage:6250 V/era: plasmid concentration: 0.5 ~tg.
regards the Bacillus genus, only B. thuringiensis and B. cereus intact cells had been successfully electroporated [4,5]. In optimizing the method for B. subtilis PB1424, we determined the effect of several variables on transformation efficiency such as growth phase and cell concentration, ionic su~ngth of electroporation buffer, pulse amplitude and duration, D N A concentration and size. The best electroporation efficiency (2.8 x 104) is obtained using exponentially growing cells, frozen and thawed, in PEB buffer, electroporated at 25 laF and 6250 V / c m , with 0.5 lag of the small plasmid pVEI8 (4.6 kb).
REFERENCES It] Bennett, P.M. and Grinsted, J. (1984) Methods in Microbio|ogy, Vol. 17, AcademicPress, London. [2] Luchansky, J.B,, Muriana, P.M. and Klaenhammer. T.R. (1988) Mol. Mierobiol. 2. 637-646. [3] Shivarova,N.. Former, W., Jacob, H.E. and Grigorova, R. (1983) Z. AUg.Mikrobiol. 23. 595-599. [4] Bone, E.J, and Eliot, DJ. 0989) FEMS Microbiol. Leo. S8. 171-178. lSl Belliveao, B.H, and Trevors, J.T. (1989) Appl. Environ. Microbiol. 55,1649-1652. 161 Maniatis, T., Frltseh, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. [7] De Rossi, E., Bri$idi. P., Riccardi. G, and Maneuzzl. D. (1989) Corr. Mierobiol. 19.13+19, [81 HorinouchL S. and Weisblum, B. (1982) J. Bacteriol. 150, 815-825. 19] Ehrlich, S.D. (1978) Ptoc. Natl. Acad. Sci. U.S.A. 75, 1433-1436,
