182
CLONINGANDRECOMBINANTDNA
[12]
DNA probes (e.g., rDNA or the gene for the translation elongation factor ltx24), hybridization signals are seen with DNA of all Saccharomyces species and even other yeast genera. Acknowledgments We thank Sue Klapholz, Jfirg Gafner, and Hans Hegemann for useful comments and Valerie' Lui for typing the manuscript. This work was supported by the Swiss National Science Foundation and by the Deutsche Forschungsgemeinschafl (SFB 272). 24E Schirmaier and P. Philippsen, EMBO Z 3, 3311 (1984).
[1 2] H i g h - E f f i c i e n c y T r a n s f o r m a t i o n Electroporation
of Yeast by
By DA~IF.L M. BECKERand LEONARDGUARE~TE Introduction A prerequisite for molecular biological manipulation of any organism is a reliable and efficient means for introducing exogenous DNA into the cell. Yet each of the techniques in general use for transforming yeast, namely, lithium acetate transformation ~ and spheroplast transformation, 2 suffers from significant limitations. Lithium acetate transformation, although relatively fast and simple, provides only a low efficiency of DNA transfer ( - l0 s colonies//zg of episomal plasmid). Spheroplast transformation, while more efficient ( - 1-5 X 104 colonies//zg), is complicated and time consuming. We present here a method for transforming yeast by electroporation that is extremely simple and an order of magnitude more efficient than spheroplast transformation. A number of groups have attempted previously to introduce macromolecules into yeast by dectroporation, 3-5 but efforts to introduce plasraids have failed to yield transformation efficiencies greater than approxiH. Ito, Y. Fukada, K. Murata, and A. Kimura, J. Bacteriol. 153, 163 (1983). 2 A. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978). 3 H. Hashimoto, H. Morikawa, Y. Yamada, and A. Kimura, Appl. Microbiol. Biotechnol. 21, 336 (1985). 4 I. Karube, E. Tamiya, and H. Matsuoka, FEBSLett. 182, 90 (1985). s I. Uno, K. Fukami, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188
(1988). METHODS IN ENZYMOLOGY, VOL. 194
~ t O 1991 by Academic Pica, Inc. All rightmofrepfoduefion in any form re~rved.
[ 12]
TRANSFORMATION OF YEASTBYELECTROPORATION
183
mately 1.5 X 103/#g of DNA. The low efficiency is surprising, as 35-75% of yeast cells in a population can be demonstrated to take up macromolecules after an electric pulse.6 This paradox s~_l~ecstedto us that the failure might be attributable, at least in part, to inadequate stabilizarion of the membrane of cells that had in fact been permeabilized and transformed. Further, recent publication of a protocol for high-efficiency electroporarion of bacteria7 suggested solutions to other problems inherent in the electroporation of organisms as small as yeast. We have, therefore, designed our protocol by adapting the principles of bacterial electroporarion to transformarion of Saccharomyces cerevisiae, taking care, in addition, to provide continuous osmotic support of the electrically compromised cells.
Preparation of Electrocompetent Cells 1. Inoculate 500 ml YPD in a 2-liter flask with an aliquot from an overnight culture. Grow with vigorous shaking at 30 ° to an OD60o of 1.3-1.5. We usually start two to four cultures simultaneously with inocula of different volumes the night before the transformation, in order to guarantee one culture at the proper density at a reasonable rime the next morning. With the strain that we use most commonly, BWGI-Ta (MATa leu2-3,112 ura3-52 hi84-519 adel-lO0), an optical density of 1.3-1.5 at 600 nm corresponds to a density of approximately 1 X 108 cells/ml. The growth phase of the culture is extremely important: testing parallel cultures at optical densities varying over a 5-fold range (0.245 to 1.3), we observe a 60-fold increase in transformation efficiency (6.5 X 103//~g to 3.8 X 10s//~g). The overnight culture need not be fresh, and the culture can also be started with an inoculum directly from a plate. 2. Divide the culture into two 250-ml centrifuge bottles and spin at 5000 rpm for 5 rain at 4 ° in a Sorvall GSA rotor. Discard the supernatant. This and the subsequent centrifugations achieve two purposes: they concentrate the cells 500- to 1000-fold and reduce the conductivity of the culture dramatically. The exact volumes, rotors, and centrifugation rimes are not critical as long as both of the goals are met. It is important, however, to keep the culture and all solutions cold. 3. Resuspend in a total of 500 ml ice-cold sterile water. If the culture is divided in half, as suggested, resuspend each bottle in 6 j. C. Weaver, G. I. Harrison, J. G. Bliss, J. R. Mourant, and K. T. Powell, F E B S Lett. 229, 30 (1988). 7 W. J. Dower, J. F. Miller, and C. W. Ragsdale, NucleicAcids Res. 16, 6127 (1988).
184
CLONINGANDRBCOMBmANT DNA
[ 12]
250 ml. Resuspension is most easily accomplished by vortexing with about 100 ml, then adding the remainder of the water and shaking the bottle. 4. Centrifuge at 5000 rpm for 5 rain at 4 ° and discard the supernatant. 5. Resuspend in a total of 250 ml ice-cold sterile water. At this step, we pool the two aliquots (125 ml each) of the original culture into a single bottle. 6. Centrifuge as above, and discard the supernatant. 7. Resuspend in 20 ml ice-cold 1 M sorbitol (182 g/liter in sterile, distilled water). Transfer to a chilled 30-ml centrifuge tube. We vortex initially, then complete the resuspension by pipetting up and down in the sterile 25-ml pipette used to transfer the culture to the smaller centrifuge tube. 8. Spin at 5000 rpm for 5 min at 4 ° in a Sorvall SS34 rotor. Discard the supematant. 9. Resuspend by adding 0.5 ml ice-cold 1 M sorbitol. Store on ice. We resuspend by pipetting up and down in a l-m1 serologic pipette. The final volume varies from about 1 to 1.5 ml. Electroporation 1. Aliquot 40 ~1 of yeast suspension per transformation to a sterile Eppendorf tube. Add - 100 ng DNA in at most 5 ~1 to the tube, mix gently, and incubate on ice about 5 rain. Increasing the volume of yeast from 40 to 160 ~1 has no effect on the number of transformants recovered, suggesting that the number of transformation events under these conditions is not limited by the number of electrocompetent yeast in the reaction. Further, these data provide additional evidence that the increase in efficiency observed with increasing optical density is a function not of cell density per se, but of the growth phase of the culture. Note, too, that none of the above preparative steps disrupts t h e yeast cell wall; electroporation of spheroplasts prepared with Glusulase and plated in top agar provides efficiencies no better than those obtained with intact yeast with the procedure described here. The DNA should be in a low ionic strength buffer such as TE (10 m M Tris-HC1, 1 mM EDTA, pH 8.0) and must be in as small a volume as possible. Addition of carrier DNA reduces the transformation efficiency. Incubation time can be varied to convenience. 2. Transfer to a cold 0.2-cm sterile electroporation cuvette. Tap to the bottom. 3. Pulse at 1.5 kV, 25/~F, 200 fl. We use a commercial apparatus with commercially available cuvettes [Bio Pad Gene Pulser with Pulse Controller, with Bio Rad 0.2-cm cuvettes (Richmond, CA)]. This device discharges an exponential pulse through the
[ 12]
TRANSFORMATION OF YEAST BY ELECTROPORATION
185
cuvette. With electrodes separated by 0.2 em and the initial voltage set to 1.5 kV, the initial field strength E. is 7.5 kV/em. With the resi__smnceof the yeast/DNA mixture high, the time constant for decay is determined almost exclusively by the capacitance and resistance set in parallel, and it varies slightly around the theoretical value of 5 msee. We have not tested devices from other manufacturers, but we expect that any device which can reproduce these electric parameters should suffice. 4. Immediately add 1 ml cold 1 M sorbitol to the cuvette. Using a sterile Pasteur pipette, gently mix the contents of the cuvette and transfer to a culture tube. We have found that including other components with the sorbitol, such as diluted YPD or CaC12, reduces the efficiency. Plating 1. Plate aliquots of the transformation by spreading on selective plates containing 1 M sorbitol. No incubation is required after resuspension and transfer of the reaction from the cuvette; we plate as soon as possible after electroporation. The manner in which the transformed yeast are plated is crucial to the transformation efficiency. The data in Table I are from an experiment in which two transformations (A and B) are performed in parallel using aliquots from the same preparation of electrocompetent cells. Each aliquot is transformed with 100 ng of plasmid, and portions of the transformed cells are plated in duplicate using a variety of techniques. Numbers represent averages of duplicate platings and are expressed as transformants per microgram. TABLE I REPLICAPLATING Composition Top afar lacking sorbitol on a plate lacking sorbitol Top agar lacking sorbitol on a plate containing sorbitol Top agar containing sorbitol on a plate containing sorbitol Spread on a plate lacking sorbitol Spread on a plate containing sorbitol
Transformants A 1.5 X 102 B 6 . 0 × 102 A 1.1X 103 B 2.9 X 103 A 1.3 X 105 B 4.5 X 104 A 2.0 X 104 B 2 . 6 X 104 A > 3 . 0 X 10s~ B > 3 . 0 X l0 s`
a The density of plating preclud~ mm'e a~urate quantit~tion.
186
CLONINOANDRECOMBINANTDNA
[ 12]
Transformation of yeast by this procedure involves a minimum of manipulations. Preparation of cells in advance of electroporation involves four 5-rain centrifugations. There is minimal preincubation with DNA and no carder nucleic acid, and the pulse itself takes only a moment. Subsequent outgrowth is not required, and plating by spreading is sufficient to provide maximal efficiency. Using this procedure, we routinely obtain cfficiencies of 2-5 X 10s colonies//tg using episomal plasmids of various sizes. This is a 10-fold higher efficiency than that which we observe, with the same plasmids, using spheroplast transformation. Although BWGI-Ta and its derivatives arc known to transform relatively well by all techniques, comparable results have been obtained using other strains, including pcrites. We note, however, decreasing transformation efficiencies with increases of input DNA above 100 ng, limiting the utility of this protocol in certain circumstances, so we include below the protocols used in our laboratory for transformation of yeast by lithium acetate (based on Ref. 1) and by spheroplasting (based on Ref. 2). Lithium Acetate Transformation 1. Inoculate 100 ml YPD and grow with vigorous shaking at 30 ° to an OD6oo of 1.0-2.0. 2. Spin at 3000 rpm for 5 min. Resuspend in 5 ml TE (10 m M Tris-HCl, pH 7.5, l m M EDTA). Repeat the spin twice more, resuspending first in 5 ml of TE containing 100 m M lithium acetate, then in 1 ml TE/lithium acetate. 3. Shake at 30 ° for 60 min. 4. Add 400/lg carrier (calf thymus) DNA. 5. Aliquot 100/J1 per transformation to sterile Eppendorf tube. 6. Add 1/lg transforming DNA. 7. Incubate for 30 rain, 30 °. 8. Add 700/ll PEG solution [35% (w/v) PEG 4000, 100 mMlithium acetate, TE, pH 7.5]. 9. Incubate for 50 min at 30 °. 10. Heat shock for 5 rain at 42 °. 11. Pellet 4 sec. Aspirate supernatant. Wash with 500 pl TE. Resuspend in 100/d TE. Plate on selective media. Spheroplast Transformation 1. Inoculate 100 ml YPD in a 2-liter flask with an aliquot from an overnight culture. Grow with vigorous shaking at 30 ° to an ODeo of 0.6-0.8.
[13]
CONJUOATION B E T W E E N BACTERIA A N D YEAST
187
2. Spin at 3000 rpm for 5 rain at room temperature. Decant supernatant, and msuspend in 5 ml of 1.2 M sorbitol. Spin again, rcsuspending in 5 ml of 1.2 M sorbitol. 3. Add 50/~1 Glusulasc (Du Pont, Wilmington, DE). 4. Incubate at 30 ° for l hr, swirling gently every l0 min. It is possible at this point to make a visual check of the effectiveness of sphcroplasting by diluting a small volume of Glusulasc-trcatcd cells ( - 5 #l) into a drop of 5% SDS on a microscope slide and observing the formation of ghosts. We routinely proceed without this check. 5. Rinse sphcroplasts 4 times with 5 ml of 1.2 M sorbitol, spinning at 3000 rpm, room temperature, each time. 6. Rcsuspend the final pellet in 0.6 ml STC (1 M sorbitol, l0 mM Tris-HCl, pH 7.5, 10 mMCaCl2). 7. Aliquot 100/~l per transformation tube (Falcon 2059). Add 1 #g transforming DNA and 9 #g carrier (calf thymus) DNA. 8. Incubate for 5 min at room temperature. 9. Add 4 ml PEG solution [20O/o (w/v) PEG 4000, l0 rnM CaCl2, l0 rnM Tris-HCl, pH 7.4]. Mix gently. 10. Incubate for l0 min at room temperature. 11. Pellet in a tabletop centrifuge for 5 min at room temperature. Rcsuspend in 150/~1 SOS (l M sorbitol, 6.5 m M CaCl2, ~ strength YPD, strength auxotrophy supplement). 12. Incubate at 30 ° for 20-60 rain. 13. Plate in 5-6 ml regeneration top agar (l X SD, 1.2 M sorbitol, 2% agar, auxotrophy supplements) on a plate containing 1.2 M sorbitol.
Acknowledgments This work was supportedby grantsfrom the NationalInstitutesof Health (toL.G.)and by a postdoctoralfellowshipfrom the American Cancer Society(to D.M.B.).
[13] Transmission of Plasmid D N A to Yeast by Conjugation with Bacteria By J A C K A . H E I N E M A N N a n d G E O R G E F. S P R A G U E , JR. Introduction It has been known since the 1940s that genetic information can be transmitted from one bacterial cell to another by a process t~'mod conjugation. ~ The donor cell in a conjugation reaction harbors a conjugative METHOD~ IN ENZYMOLOGY,VOL. 194
COlB~6~© 1991by Am&m~ Pn~, Inc. Anri~mofre~xlu~/oaln my fonnm~ved.
CLONINGANDRECOMBINANTDNA
[12]
DNA probes (e.g., rDNA or the gene for the translation elongation factor ltx24), hybridization signals are seen with DNA of all Saccharomyces species and even other yeast genera. Acknowledgments We thank Sue Klapholz, Jfirg Gafner, and Hans Hegemann for useful comments and Valerie' Lui for typing the manuscript. This work was supported by the Swiss National Science Foundation and by the Deutsche Forschungsgemeinschafl (SFB 272). 24E Schirmaier and P. Philippsen, EMBO Z 3, 3311 (1984).
[1 2] H i g h - E f f i c i e n c y T r a n s f o r m a t i o n Electroporation
of Yeast by
By DA~IF.L M. BECKERand LEONARDGUARE~TE Introduction A prerequisite for molecular biological manipulation of any organism is a reliable and efficient means for introducing exogenous DNA into the cell. Yet each of the techniques in general use for transforming yeast, namely, lithium acetate transformation ~ and spheroplast transformation, 2 suffers from significant limitations. Lithium acetate transformation, although relatively fast and simple, provides only a low efficiency of DNA transfer ( - l0 s colonies//zg of episomal plasmid). Spheroplast transformation, while more efficient ( - 1-5 X 104 colonies//zg), is complicated and time consuming. We present here a method for transforming yeast by electroporation that is extremely simple and an order of magnitude more efficient than spheroplast transformation. A number of groups have attempted previously to introduce macromolecules into yeast by dectroporation, 3-5 but efforts to introduce plasraids have failed to yield transformation efficiencies greater than approxiH. Ito, Y. Fukada, K. Murata, and A. Kimura, J. Bacteriol. 153, 163 (1983). 2 A. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978). 3 H. Hashimoto, H. Morikawa, Y. Yamada, and A. Kimura, Appl. Microbiol. Biotechnol. 21, 336 (1985). 4 I. Karube, E. Tamiya, and H. Matsuoka, FEBSLett. 182, 90 (1985). s I. Uno, K. Fukami, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188
(1988). METHODS IN ENZYMOLOGY, VOL. 194
~ t O 1991 by Academic Pica, Inc. All rightmofrepfoduefion in any form re~rved.
[ 12]
TRANSFORMATION OF YEASTBYELECTROPORATION
183
mately 1.5 X 103/#g of DNA. The low efficiency is surprising, as 35-75% of yeast cells in a population can be demonstrated to take up macromolecules after an electric pulse.6 This paradox s~_l~ecstedto us that the failure might be attributable, at least in part, to inadequate stabilizarion of the membrane of cells that had in fact been permeabilized and transformed. Further, recent publication of a protocol for high-efficiency electroporarion of bacteria7 suggested solutions to other problems inherent in the electroporation of organisms as small as yeast. We have, therefore, designed our protocol by adapting the principles of bacterial electroporarion to transformarion of Saccharomyces cerevisiae, taking care, in addition, to provide continuous osmotic support of the electrically compromised cells.
Preparation of Electrocompetent Cells 1. Inoculate 500 ml YPD in a 2-liter flask with an aliquot from an overnight culture. Grow with vigorous shaking at 30 ° to an OD60o of 1.3-1.5. We usually start two to four cultures simultaneously with inocula of different volumes the night before the transformation, in order to guarantee one culture at the proper density at a reasonable rime the next morning. With the strain that we use most commonly, BWGI-Ta (MATa leu2-3,112 ura3-52 hi84-519 adel-lO0), an optical density of 1.3-1.5 at 600 nm corresponds to a density of approximately 1 X 108 cells/ml. The growth phase of the culture is extremely important: testing parallel cultures at optical densities varying over a 5-fold range (0.245 to 1.3), we observe a 60-fold increase in transformation efficiency (6.5 X 103//~g to 3.8 X 10s//~g). The overnight culture need not be fresh, and the culture can also be started with an inoculum directly from a plate. 2. Divide the culture into two 250-ml centrifuge bottles and spin at 5000 rpm for 5 rain at 4 ° in a Sorvall GSA rotor. Discard the supernatant. This and the subsequent centrifugations achieve two purposes: they concentrate the cells 500- to 1000-fold and reduce the conductivity of the culture dramatically. The exact volumes, rotors, and centrifugation rimes are not critical as long as both of the goals are met. It is important, however, to keep the culture and all solutions cold. 3. Resuspend in a total of 500 ml ice-cold sterile water. If the culture is divided in half, as suggested, resuspend each bottle in 6 j. C. Weaver, G. I. Harrison, J. G. Bliss, J. R. Mourant, and K. T. Powell, F E B S Lett. 229, 30 (1988). 7 W. J. Dower, J. F. Miller, and C. W. Ragsdale, NucleicAcids Res. 16, 6127 (1988).
184
CLONINGANDRBCOMBmANT DNA
[ 12]
250 ml. Resuspension is most easily accomplished by vortexing with about 100 ml, then adding the remainder of the water and shaking the bottle. 4. Centrifuge at 5000 rpm for 5 rain at 4 ° and discard the supernatant. 5. Resuspend in a total of 250 ml ice-cold sterile water. At this step, we pool the two aliquots (125 ml each) of the original culture into a single bottle. 6. Centrifuge as above, and discard the supernatant. 7. Resuspend in 20 ml ice-cold 1 M sorbitol (182 g/liter in sterile, distilled water). Transfer to a chilled 30-ml centrifuge tube. We vortex initially, then complete the resuspension by pipetting up and down in the sterile 25-ml pipette used to transfer the culture to the smaller centrifuge tube. 8. Spin at 5000 rpm for 5 min at 4 ° in a Sorvall SS34 rotor. Discard the supematant. 9. Resuspend by adding 0.5 ml ice-cold 1 M sorbitol. Store on ice. We resuspend by pipetting up and down in a l-m1 serologic pipette. The final volume varies from about 1 to 1.5 ml. Electroporation 1. Aliquot 40 ~1 of yeast suspension per transformation to a sterile Eppendorf tube. Add - 100 ng DNA in at most 5 ~1 to the tube, mix gently, and incubate on ice about 5 rain. Increasing the volume of yeast from 40 to 160 ~1 has no effect on the number of transformants recovered, suggesting that the number of transformation events under these conditions is not limited by the number of electrocompetent yeast in the reaction. Further, these data provide additional evidence that the increase in efficiency observed with increasing optical density is a function not of cell density per se, but of the growth phase of the culture. Note, too, that none of the above preparative steps disrupts t h e yeast cell wall; electroporation of spheroplasts prepared with Glusulase and plated in top agar provides efficiencies no better than those obtained with intact yeast with the procedure described here. The DNA should be in a low ionic strength buffer such as TE (10 m M Tris-HC1, 1 mM EDTA, pH 8.0) and must be in as small a volume as possible. Addition of carrier DNA reduces the transformation efficiency. Incubation time can be varied to convenience. 2. Transfer to a cold 0.2-cm sterile electroporation cuvette. Tap to the bottom. 3. Pulse at 1.5 kV, 25/~F, 200 fl. We use a commercial apparatus with commercially available cuvettes [Bio Pad Gene Pulser with Pulse Controller, with Bio Rad 0.2-cm cuvettes (Richmond, CA)]. This device discharges an exponential pulse through the
[ 12]
TRANSFORMATION OF YEAST BY ELECTROPORATION
185
cuvette. With electrodes separated by 0.2 em and the initial voltage set to 1.5 kV, the initial field strength E. is 7.5 kV/em. With the resi__smnceof the yeast/DNA mixture high, the time constant for decay is determined almost exclusively by the capacitance and resistance set in parallel, and it varies slightly around the theoretical value of 5 msee. We have not tested devices from other manufacturers, but we expect that any device which can reproduce these electric parameters should suffice. 4. Immediately add 1 ml cold 1 M sorbitol to the cuvette. Using a sterile Pasteur pipette, gently mix the contents of the cuvette and transfer to a culture tube. We have found that including other components with the sorbitol, such as diluted YPD or CaC12, reduces the efficiency. Plating 1. Plate aliquots of the transformation by spreading on selective plates containing 1 M sorbitol. No incubation is required after resuspension and transfer of the reaction from the cuvette; we plate as soon as possible after electroporation. The manner in which the transformed yeast are plated is crucial to the transformation efficiency. The data in Table I are from an experiment in which two transformations (A and B) are performed in parallel using aliquots from the same preparation of electrocompetent cells. Each aliquot is transformed with 100 ng of plasmid, and portions of the transformed cells are plated in duplicate using a variety of techniques. Numbers represent averages of duplicate platings and are expressed as transformants per microgram. TABLE I REPLICAPLATING Composition Top afar lacking sorbitol on a plate lacking sorbitol Top agar lacking sorbitol on a plate containing sorbitol Top agar containing sorbitol on a plate containing sorbitol Spread on a plate lacking sorbitol Spread on a plate containing sorbitol
Transformants A 1.5 X 102 B 6 . 0 × 102 A 1.1X 103 B 2.9 X 103 A 1.3 X 105 B 4.5 X 104 A 2.0 X 104 B 2 . 6 X 104 A > 3 . 0 X 10s~ B > 3 . 0 X l0 s`
a The density of plating preclud~ mm'e a~urate quantit~tion.
186
CLONINOANDRECOMBINANTDNA
[ 12]
Transformation of yeast by this procedure involves a minimum of manipulations. Preparation of cells in advance of electroporation involves four 5-rain centrifugations. There is minimal preincubation with DNA and no carder nucleic acid, and the pulse itself takes only a moment. Subsequent outgrowth is not required, and plating by spreading is sufficient to provide maximal efficiency. Using this procedure, we routinely obtain cfficiencies of 2-5 X 10s colonies//tg using episomal plasmids of various sizes. This is a 10-fold higher efficiency than that which we observe, with the same plasmids, using spheroplast transformation. Although BWGI-Ta and its derivatives arc known to transform relatively well by all techniques, comparable results have been obtained using other strains, including pcrites. We note, however, decreasing transformation efficiencies with increases of input DNA above 100 ng, limiting the utility of this protocol in certain circumstances, so we include below the protocols used in our laboratory for transformation of yeast by lithium acetate (based on Ref. 1) and by spheroplasting (based on Ref. 2). Lithium Acetate Transformation 1. Inoculate 100 ml YPD and grow with vigorous shaking at 30 ° to an OD6oo of 1.0-2.0. 2. Spin at 3000 rpm for 5 min. Resuspend in 5 ml TE (10 m M Tris-HCl, pH 7.5, l m M EDTA). Repeat the spin twice more, resuspending first in 5 ml of TE containing 100 m M lithium acetate, then in 1 ml TE/lithium acetate. 3. Shake at 30 ° for 60 min. 4. Add 400/lg carrier (calf thymus) DNA. 5. Aliquot 100/J1 per transformation to sterile Eppendorf tube. 6. Add 1/lg transforming DNA. 7. Incubate for 30 rain, 30 °. 8. Add 700/ll PEG solution [35% (w/v) PEG 4000, 100 mMlithium acetate, TE, pH 7.5]. 9. Incubate for 50 min at 30 °. 10. Heat shock for 5 rain at 42 °. 11. Pellet 4 sec. Aspirate supernatant. Wash with 500 pl TE. Resuspend in 100/d TE. Plate on selective media. Spheroplast Transformation 1. Inoculate 100 ml YPD in a 2-liter flask with an aliquot from an overnight culture. Grow with vigorous shaking at 30 ° to an ODeo of 0.6-0.8.
[13]
CONJUOATION B E T W E E N BACTERIA A N D YEAST
187
2. Spin at 3000 rpm for 5 rain at room temperature. Decant supernatant, and msuspend in 5 ml of 1.2 M sorbitol. Spin again, rcsuspending in 5 ml of 1.2 M sorbitol. 3. Add 50/~1 Glusulasc (Du Pont, Wilmington, DE). 4. Incubate at 30 ° for l hr, swirling gently every l0 min. It is possible at this point to make a visual check of the effectiveness of sphcroplasting by diluting a small volume of Glusulasc-trcatcd cells ( - 5 #l) into a drop of 5% SDS on a microscope slide and observing the formation of ghosts. We routinely proceed without this check. 5. Rinse sphcroplasts 4 times with 5 ml of 1.2 M sorbitol, spinning at 3000 rpm, room temperature, each time. 6. Rcsuspend the final pellet in 0.6 ml STC (1 M sorbitol, l0 mM Tris-HCl, pH 7.5, 10 mMCaCl2). 7. Aliquot 100/~l per transformation tube (Falcon 2059). Add 1 #g transforming DNA and 9 #g carrier (calf thymus) DNA. 8. Incubate for 5 min at room temperature. 9. Add 4 ml PEG solution [20O/o (w/v) PEG 4000, l0 rnM CaCl2, l0 rnM Tris-HCl, pH 7.4]. Mix gently. 10. Incubate for l0 min at room temperature. 11. Pellet in a tabletop centrifuge for 5 min at room temperature. Rcsuspend in 150/~1 SOS (l M sorbitol, 6.5 m M CaCl2, ~ strength YPD, strength auxotrophy supplement). 12. Incubate at 30 ° for 20-60 rain. 13. Plate in 5-6 ml regeneration top agar (l X SD, 1.2 M sorbitol, 2% agar, auxotrophy supplements) on a plate containing 1.2 M sorbitol.
Acknowledgments This work was supportedby grantsfrom the NationalInstitutesof Health (toL.G.)and by a postdoctoralfellowshipfrom the American Cancer Society(to D.M.B.).
[13] Transmission of Plasmid D N A to Yeast by Conjugation with Bacteria By J A C K A . H E I N E M A N N a n d G E O R G E F. S P R A G U E , JR. Introduction It has been known since the 1940s that genetic information can be transmitted from one bacterial cell to another by a process t~'mod conjugation. ~ The donor cell in a conjugation reaction harbors a conjugative METHOD~ IN ENZYMOLOGY,VOL. 194
COlB~6~© 1991by Am&m~ Pn~, Inc. Anri~mofre~xlu~/oaln my fonnm~ved.
