CRH/018
Letters in Applied Microbiology 1990,11, 62-64
High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230 with plasmid pGB301 S U S A ND O R N A N& * M . A . C O L L I N S * ? *Department of Food and Agricultural Microbiology, Queen's University of Belfast and ?Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5 P X , Northern Ireland Received 23 February 1990 and accepted 4 March 1990 D O R N A NS., & C O L L I N SM.A. , 1990. High efiiciency electroporation of Lactococcus laetis subsp. lactis LM0230 with plasmid pGB301. Letters in Applied Microbiology 1 1 , 6 2 4 . Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al. 1981), has been reported by McIntyre & Harlander (1989a). Improved transformation efficiencies of 102-103/pg DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b). This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc. lactis subsp. lactis.
Materials and Methods RACTERIAL STRAINS A N D GROWTH CONDITIONS
Bacillus subtilis CU403 divIV-Bl(pGB301) (M.S. Gilmore, University of Oklahoma) harbouring plasmid pGB301 (9.8 kilobase; conferring chloramphenicol and erythromycin resistance) was propagated at 37°C on Brain Heart Infusion agar (Gibco BRL) containing 5 pg/ml chloramphenicol (Sigma) and 50 pg/ml erythromycin (Sigma). Lactococcus lactis subsp. lactis LM0230 (L.L. McKay, University of Minnesota) was grown on GM17 agar (Oxoid CM785, in which lactose was replaced with 1% w/v glucose) at 30°C under 5% CO, . PLASMID D N A PREPARATION
A N D ISOLATION
Plasmid DNA was extracted from Bacillus subtilis CU403 divIV-B1 (pGB301) using the alkaline lysis procedure (Maniatis et al. 1982) with Address for correspondence:Dr M.A. Collins, Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 SPX, Northern Ireland.
modifications (Kok et al. 1984). Extracted DNA was purified by cesium chloride-ethidium bromide density centrifugation (Maniatis et al. 1982). DNA concentration was determined spectrophotometrically at an absorbance of 260nm. The method of Anderson & McKay (1983) was used for the isolation of plasmid DNA from Lc. lactis subsp. lactis. Plasmids were separated by agarose gel electrophoresis (Bio-Rad DNA Sub Cell) through a 0.6% agarose gel in TAE buffer (0.04 mol/l Trisacetate, 0.002 mol/l EDTA, pH 8.0). E L E c T R O POR A T I O N OF
SUBSP.
Lactococcus lactis
lactis
A modification of the method of van der Lelie (1989) was used to transform Lc. lactis subsp. lactis. Overnight cultures of Lc. lactis subsp. lactis grown at 30°C under 5% CO, in GM17 broth supplemented with 40 mmol/l L-threonine (Sigma) were diluted 1:12.5 in 25 ml of GM17. Cells were harvested by centrifugation at loo00 g for 10 min when the optical density (measured at 660 nm) was between 0.26 and 0.38. The cells were washed sequentially with the ice-cold solutions given in Table 1 by alternate centrifugation and resuspension.
Electroporation of Lc. l a d s Table 1. Washing solutions used for the preparation of Lactococcus lactis subsp. lactis LM0230 cells for eIectroporation with plasmid pGB301 ~
Washing solutions
Volume (ml)
Double-distilled water Double-distilled water 50 mmol/l EDTA* Double-distilled water 0.3 mol/l sucrose 0.3 molp sucrose
2.0 1 .o 1.o 1 .o 1 .o 0.2
* Cell suspensions kept on ice for 5 min. After the final resuspension in 0.3 mol/l sucrose, the cells were immediately electroporated using a Gene Pulser (Bio-Rad Laboratories, California). Electroporation was performed by a single pulse at 2500 V, 200 R, 25 ptF in a pre-chilled 2 mm cuvette using 1 pg of purified plasmid DNA. The cell suspension was diluted immediately by the addition of 5 ml GSM17 (GM17 containing 1% w/v sucrose) broth supplemented with 25 ng of chloramphenicol and incubated for 2 h at 30°C under 5% CO, before being plated on GSM17 agar supplemented with 2.5 pg of chloramphenicol. Appropriate controls confirmed the absence of transformants if eithcr the electric pulse or the plasmid DNA was omitted. Transformation was confirmed by double antibiotic selection (chloramphenicol and erythromycin) and plasmid analyses.
Results and Discussion
Transformation efficiencies achieved for the electroporation of plasmid pGB301 into Lc.
lactis subsp. lactis LM0230 are shown in Table 2. Plasmid analyses of 20 putative transformants selected randomly from each trial revealed the presence of a molecular species equivalent in size to plasmid pGB301, and which was absent from the wild-type LM0230 strain. The conditions employed in this study permitted the recovery of transformants at high efficiencies, comparable to those obtained by Powell et al. (1988) but without the need for lysozyme pretreatment of cells. However, the reproducibly high transformation efficiencies of cell suspensions prepared on separate days was probably the most significant finding in this study and is in marked contrast to the highly variable efficiencies of electroporation-mediated transformation reported recently for Lc. lacfis subsp. lactis LM0230 (McIntyre & Harlander 1989a, 1989b). It is unlikely that this difference was due to the age and growth phase of cells subjected to electroporation as these parameters are unlikely to exert much effect at the cell concentrations and electroporation field strengths employed. However, McIntyre & Harlander (1989a, 1989b) reported considerably increased transformation efficiencies using late stationary phase cells at higher cell concentrations and electroporation field strengths. The reproducibility of transformation reported in this study is likely to be the result of the adoption of a thorough pre-electroporation cell washing regime from van der Lelie et al. (1989), which enabled closely , comparable electroporation time constants to be achieved for the separate experiments (Table 2). Use of this protocol may also have made a significant contribution to the high transformation efficiencies observed.
Table 2. Transformation efficiencies of Lactococcus lactis subsp. lactis LM0230 cells* electroporated with plasmid pGB301 using the Gene
Pulsert Trial no. 1 2
3
Time constant (ms) 3.5 3.7 3.6
63
Viable cell count (10' cfu/ml)$
Transformation efficiency (transformantslpg of DNA)
8.2
4.95 1.3
* Cells grown in GM17 supplemented with 40 mmol/l L-threonine.
t Cells porated at 2500 V ( E = 12.5 kV/cm), 200 0, 25 pF in the presence of 1 pg of plasmid DNA. $ Viable cell count prior to electroporation.
64
Susan Dornan and M . A . Collins
References ANDERSON,D.G. & MCKAY, L.L. 1983 Simple and rapid method for isolating large plasmid D N A from lactic streptococci. Applied and Environmental Microbiology 46, 549-552. BEHNKE,D., GILMORE, M.S. & FERRETTI, J.J. 1981 Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamycin/kanamycin resistance determinant. Molecular General Genetics 182,414-421. KOK, J., VAN DER VOSSEN,J.M.B.M. & VENEMA,G . 1984 Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Applied and Environmental Microbiology 48,726731. MANIATIS,T., FRITSCH, E.F. & SAMBROOK, J. 1982 Molecular Cloning. New York: Cold Spring Harbor Laboratory.
MCINTYRE,D.A. & HARLANUER, S.K. 1989a Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Applied and Environmental Microbiology 55,604-610. MCINTYRE, D.A. & HARLANDER, S.K. 1989b Improved electroporation eficiency of intact Lactococcus lactis subsp. lactis grown in defined media. Applied and Environmental Microbiology 55,2621 -2626. POWELL, I.B., ACHEN,M.G., HILLIER, A.J. & DAVIDSON, B.E. 1988 A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Applied and Enivironmental Microbiology 54,655-660. VAN DER LELIE,D., BRON,S., VENEMA, G . & OSKAM, L. 1989 Similarity of minus origin of replication and flanking region open reading frames of plasmids pUB110, pTb913 and pMV158. Nucleic Acids Research 17,7283-7294.
Letters in Applied Microbiology 1990,11, 62-64
High efficiency electroporation of Lactococcus lactis subsp. lactis LM0230 with plasmid pGB301 S U S A ND O R N A N& * M . A . C O L L I N S * ? *Department of Food and Agricultural Microbiology, Queen's University of Belfast and ?Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 5 P X , Northern Ireland Received 23 February 1990 and accepted 4 March 1990 D O R N A NS., & C O L L I N SM.A. , 1990. High efiiciency electroporation of Lactococcus laetis subsp. lactis LM0230 with plasmid pGB301. Letters in Applied Microbiology 1 1 , 6 2 4 . Electroporation-mediated transformation of Lactococcus lactis with plasmid pGB301, a 9.8 kilobase pair vector (Behnke et al. 1981), has been reported by McIntyre & Harlander (1989a). Improved transformation efficiencies of 102-103/pg DNA were achieved by altering the conditions under which the bacteria were grown prior to electroporation (McIntyre & Harlander 1989b). This present investigation sought to improve still further transformation efficiencies in order to provide a reliable high frequency transformation system for Lc. lactis subsp. lactis.
Materials and Methods RACTERIAL STRAINS A N D GROWTH CONDITIONS
Bacillus subtilis CU403 divIV-Bl(pGB301) (M.S. Gilmore, University of Oklahoma) harbouring plasmid pGB301 (9.8 kilobase; conferring chloramphenicol and erythromycin resistance) was propagated at 37°C on Brain Heart Infusion agar (Gibco BRL) containing 5 pg/ml chloramphenicol (Sigma) and 50 pg/ml erythromycin (Sigma). Lactococcus lactis subsp. lactis LM0230 (L.L. McKay, University of Minnesota) was grown on GM17 agar (Oxoid CM785, in which lactose was replaced with 1% w/v glucose) at 30°C under 5% CO, . PLASMID D N A PREPARATION
A N D ISOLATION
Plasmid DNA was extracted from Bacillus subtilis CU403 divIV-B1 (pGB301) using the alkaline lysis procedure (Maniatis et al. 1982) with Address for correspondence:Dr M.A. Collins, Food and Agricultural Microbiology Research Division, Department of Agriculture for Northern Ireland, Newforge Lane, Belfast BT9 SPX, Northern Ireland.
modifications (Kok et al. 1984). Extracted DNA was purified by cesium chloride-ethidium bromide density centrifugation (Maniatis et al. 1982). DNA concentration was determined spectrophotometrically at an absorbance of 260nm. The method of Anderson & McKay (1983) was used for the isolation of plasmid DNA from Lc. lactis subsp. lactis. Plasmids were separated by agarose gel electrophoresis (Bio-Rad DNA Sub Cell) through a 0.6% agarose gel in TAE buffer (0.04 mol/l Trisacetate, 0.002 mol/l EDTA, pH 8.0). E L E c T R O POR A T I O N OF
SUBSP.
Lactococcus lactis
lactis
A modification of the method of van der Lelie (1989) was used to transform Lc. lactis subsp. lactis. Overnight cultures of Lc. lactis subsp. lactis grown at 30°C under 5% CO, in GM17 broth supplemented with 40 mmol/l L-threonine (Sigma) were diluted 1:12.5 in 25 ml of GM17. Cells were harvested by centrifugation at loo00 g for 10 min when the optical density (measured at 660 nm) was between 0.26 and 0.38. The cells were washed sequentially with the ice-cold solutions given in Table 1 by alternate centrifugation and resuspension.
Electroporation of Lc. l a d s Table 1. Washing solutions used for the preparation of Lactococcus lactis subsp. lactis LM0230 cells for eIectroporation with plasmid pGB301 ~
Washing solutions
Volume (ml)
Double-distilled water Double-distilled water 50 mmol/l EDTA* Double-distilled water 0.3 mol/l sucrose 0.3 molp sucrose
2.0 1 .o 1.o 1 .o 1 .o 0.2
* Cell suspensions kept on ice for 5 min. After the final resuspension in 0.3 mol/l sucrose, the cells were immediately electroporated using a Gene Pulser (Bio-Rad Laboratories, California). Electroporation was performed by a single pulse at 2500 V, 200 R, 25 ptF in a pre-chilled 2 mm cuvette using 1 pg of purified plasmid DNA. The cell suspension was diluted immediately by the addition of 5 ml GSM17 (GM17 containing 1% w/v sucrose) broth supplemented with 25 ng of chloramphenicol and incubated for 2 h at 30°C under 5% CO, before being plated on GSM17 agar supplemented with 2.5 pg of chloramphenicol. Appropriate controls confirmed the absence of transformants if eithcr the electric pulse or the plasmid DNA was omitted. Transformation was confirmed by double antibiotic selection (chloramphenicol and erythromycin) and plasmid analyses.
Results and Discussion
Transformation efficiencies achieved for the electroporation of plasmid pGB301 into Lc.
lactis subsp. lactis LM0230 are shown in Table 2. Plasmid analyses of 20 putative transformants selected randomly from each trial revealed the presence of a molecular species equivalent in size to plasmid pGB301, and which was absent from the wild-type LM0230 strain. The conditions employed in this study permitted the recovery of transformants at high efficiencies, comparable to those obtained by Powell et al. (1988) but without the need for lysozyme pretreatment of cells. However, the reproducibly high transformation efficiencies of cell suspensions prepared on separate days was probably the most significant finding in this study and is in marked contrast to the highly variable efficiencies of electroporation-mediated transformation reported recently for Lc. lacfis subsp. lactis LM0230 (McIntyre & Harlander 1989a, 1989b). It is unlikely that this difference was due to the age and growth phase of cells subjected to electroporation as these parameters are unlikely to exert much effect at the cell concentrations and electroporation field strengths employed. However, McIntyre & Harlander (1989a, 1989b) reported considerably increased transformation efficiencies using late stationary phase cells at higher cell concentrations and electroporation field strengths. The reproducibility of transformation reported in this study is likely to be the result of the adoption of a thorough pre-electroporation cell washing regime from van der Lelie et al. (1989), which enabled closely , comparable electroporation time constants to be achieved for the separate experiments (Table 2). Use of this protocol may also have made a significant contribution to the high transformation efficiencies observed.
Table 2. Transformation efficiencies of Lactococcus lactis subsp. lactis LM0230 cells* electroporated with plasmid pGB301 using the Gene
Pulsert Trial no. 1 2
3
Time constant (ms) 3.5 3.7 3.6
63
Viable cell count (10' cfu/ml)$
Transformation efficiency (transformantslpg of DNA)
8.2
4.95 1.3
* Cells grown in GM17 supplemented with 40 mmol/l L-threonine.
t Cells porated at 2500 V ( E = 12.5 kV/cm), 200 0, 25 pF in the presence of 1 pg of plasmid DNA. $ Viable cell count prior to electroporation.
64
Susan Dornan and M . A . Collins
References ANDERSON,D.G. & MCKAY, L.L. 1983 Simple and rapid method for isolating large plasmid D N A from lactic streptococci. Applied and Environmental Microbiology 46, 549-552. BEHNKE,D., GILMORE, M.S. & FERRETTI, J.J. 1981 Plasmid pGB301, a new multiple resistance streptococcal cloning vehicle and its use in cloning of a gentamycin/kanamycin resistance determinant. Molecular General Genetics 182,414-421. KOK, J., VAN DER VOSSEN,J.M.B.M. & VENEMA,G . 1984 Construction of plasmid cloning vectors for lactic streptococci which also replicate in Bacillus subtilis and Escherichia coli. Applied and Environmental Microbiology 48,726731. MANIATIS,T., FRITSCH, E.F. & SAMBROOK, J. 1982 Molecular Cloning. New York: Cold Spring Harbor Laboratory.
MCINTYRE,D.A. & HARLANUER, S.K. 1989a Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Applied and Environmental Microbiology 55,604-610. MCINTYRE, D.A. & HARLANDER, S.K. 1989b Improved electroporation eficiency of intact Lactococcus lactis subsp. lactis grown in defined media. Applied and Environmental Microbiology 55,2621 -2626. POWELL, I.B., ACHEN,M.G., HILLIER, A.J. & DAVIDSON, B.E. 1988 A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Applied and Enivironmental Microbiology 54,655-660. VAN DER LELIE,D., BRON,S., VENEMA, G . & OSKAM, L. 1989 Similarity of minus origin of replication and flanking region open reading frames of plasmids pUB110, pTb913 and pMV158. Nucleic Acids Research 17,7283-7294.
