[4]
PLASMID TRANSFORMATION
63
[4] P l a s m i d T r a n s f o r m a t i o n o f E s c h e r i c h i a coli and Other Bacteria B y DOUGLAS HANAHAN, JOEL JESSEE,
and FREDRIC R. BLOOM
I. Introduction The discoveries that phage 1 and plasmid 2 DNAs could be transferred directly into bacterial cells following a competence induction process involving treatment with calcium chloride at low temperatures set the stage for molecular cloning of recombinant DNAs. The technique of DNA transformation has become important in virtually all aspects of molecular genetics. Escherichia coli has developed into a universal host organism both for molecular cloning of DNA and for a diverse set of assays involving clones genes. Transformation of other bacteria is also being applied for more specialized purposes, in addition to genetic studies of these organisms themselves, as the chapters throughout this volume testify. This chapter presents the major techniques and parameters that affect transformation of bacteria. The principal focus is on E. coli, although the basic principles for effectively transforming other gram-negative and grampositive bacteria are discussed as well. There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. There are two primary technical variations: chemical induction of competence and highvoltage electroshock treatment (electroporation). Both the characteristic of the cells being transformed and the purpose of the transformation will affect the choice of method. The second major parameter is the genetic constitution of the host strain of the organism being transformed, as there is clear evidence that a variety of genes can dramatically influence the outcome of transformation experiments. Since these genetic factors are increasingly well understood, they are discussed in some depth below. Methods for inducing competence by chemical treatments and electroshock are described, along with other relevant parameters, such as handling of bacteria for transformation purposes. Finally, application of these principles (especially electroshock treatment) to other bacteria is discussed. The E. coli transformation protocols to be presented are summarized in Table I. I M . Mandel and A. Higa, J. Mol. Biol. 53, 159 (1970). 2 S. Cohen, A. Chang, and L. Hsu, Proc. Natl. Acad. Sci. U.S.A. 69, 2110 (1972).
METHODS IN ENZYMOLOGY, VOL. 204
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
Escherichia coli AND Salmonella typhimurium
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TABLE I
Escherichia coli TRANSFORMATION METHODSa Typical Typical transformation competence efficiency (F0 (XFE) (%)
Protocol
Method
5
Calcium manganesebased (CCMB)
1-10 x 10s
1-5
MC1061, DH10B
6
TFB-based high efficiency
0.4-2 x 109
2-10
DHI, DH5, HB 101
7
FSB-based frozen competent
1-4
DH1, DH5, HBI01
8
PEG/DMSO one step
106-107
0.01-0.1
Most common cloning hosts
9
Rapid colony transformation
103-104
90%). 2b. For long-term storage, resuspend the cell pellet in a mixture of 80% SOB medium/20% redistilled glycerol, transfer the cell suspension to a 1.5 to 2-ml polypropylene tube (preferably screw-capped), chill on ice, flash freeze in a dry ice/ethanol bath, and place at - 80°. C. Reagents and Growth Media Both chemical and electroshock methods of transformation show some sensitivity to the quality of reagents used in the transformation buffers. This is particularly true for the high-efficiency protocols which employ dimethyl sulfoxide (DMSO), where it appears that organic contaminants, including oxidation products of DMSO itself, are highly inhibitory to the induction of competence for plasmid transformation. With regard to growth medium, there has also been variability noted in lots of digested casein (tryptone) and e~tracts of yeast with regard to competence induction. Besides possible effects of reagent quality, the transformation methods differ in the benefits of elevated levels (20 mM) of magnesium during cell growth prior to competence induction. The protocols using standard transformation buffer (TFB) or frozen storage buffer (FSB) and DMSO benefit from added magnesium, whereas the chemical protocol optimized for E. coli MC 1061 derivatives and the electrocompetenceprotocol require growth without added magnesium. 1. Quality and Suppliers. As a general rule ultrapure or reagent-grade chemicals should be employed, and most major suppliers are satisfactory (including Fluka, Ronkonkoma, NY; Mallinkrodt, St. Louis, MO; and Fisher, Fairlawn, N J). The quality of the water is very important for the procedures using DMSO and can impact on all of the methods. In general we recommend the use of water purified by reverse osmosis, as this procedure effectively removes not only salts but also organic contaminants, including those with similar volatility to water, which are not re-
[4]
PLASMID TRANSFORMATION
69
moved by distillation. However, it is important that the carbon filters which remove organics be replaced regularly, as there is no gauge to measure their saturation (except drastically reduced competence when it occurs). Distilled or deionized water can also be further purified in the laboratory with carbon, as described by Hanahan. 3 Dimethyl sulfoxide (DMSO) is subject to oxidation, and its oxidation products can be a major source of variability in the levels of competence induced by procedures which employ it. Note also that DMSO dissolves polystyrene, and therefore tubes composed of this material cannot be used. We recommend that DMSO be purchased in the smallest possible amounts (e.g., 100-ml bottles) at spectroscopy grade, aliquoted to fill small (0.5 ml) polypropylene tubes completely, and then stored at - 80°. A tube of DMSO is thawed, used for that day, and then discarded. Among the adequate suppliers of DMSO are Fluka, Mallinkrodt, and MCB. Similarly, the dithiothreitol (DTT) used in the TFB method (Protocol 6) is also subject to impurities which inhibit competence. One of the most reliable suppliers of DTT is Calbiochem (La Jolla, CA). Impurities in glycerol products can affect all of the competence and storage protocols used here, and it is recommended that redistilled or spectroscopy grades of glycerol be used. Among the recommended suppliers are Fluka and BRL (Gaithersburg, MD). Other variables which can influence competence induction are in the flasks and tubes used for culturing and treating the cells during transformation. Of particular concern are soap residues in glassware and surfactants used in manufacturing some brands of polypropylene tubes. It is recommended that glassware used in transformation procedures be thoroughly rinsed with distilled water, and autoclaved half full of water, to prevent deposition of surface residues (including soap). For E. coli we do not use glassware washing services but rather wash by hand without soap. Among the reliable suppliers of polypropylene tubes for transformation procedures are Falcon (Oxnard, CA) and Coming (Coming, NY). The components of the growth media can also influence transformation. We routinely use Bacto-tryptone and Bacto yeast extract. Alternative suppliers should be carefully compared, as lot variations have been reported with regard to transformation experiments. Again, reagent-grade chemicals and the purest available water are recommended. 2. Formulations of Media Used for Cell Growth and Recooery in Transformation Procedures. All the transformation protocols described herein use a common set of growth media, with the only major distinction 3 D. Hanahan, in " D N A Cloning Techniques" (D. Glover, ed.), p. 109. IRL Press, London, 1985.
70
Escherichia coli AND Salmonella typhimurium
[4]
being the presence or absence of elevated levels of magnesium. These differ from many traditional media in that the levels of sodium are low. The recovery of cells from all of these procedures is enhanced in SOC medium, which contains glucose. We recommend that these media be filter sterilized just prior to use, to remove particulate matter and any slow-growing contaminating microorganisms. Each medium is identical to or derived from a stock of SOB-Mg medium (SOB without magnesium), and their formulations are as follows. Compound Growth medium SOB-Mg Bacto-tryptone Bacto yeast extract NaCl KCI Growth medium SOB SOB-Mg medium MgC12 and MgSO4 Recovery medium SOC S O B - M g medium MgC12 and MgSO4 Glucose
Amount/liter
Final concentration
20 g 5g 0.58 g 0.19 g
2.0% 0.5% 10.0 mM 2.5 mM
1 liter 10 ml of a 2 M stock
99% 20 mM
1 liter 10 ml of a 2 M stock 10 ml of a 2 M stock
98% 20 raM 20 mM
Preparation. Combine tryptone, yeast extract, NaC1, and KCI in the purest available water. Aliquot into prerinsed glass flasks and autoclave for 30-40 min to generate SOB-Mg medium. Sterilize by filtration just prior to use for protocols requiring this medium itself. Prepare a 2 M stock solution of M g 2+ by combining 203 g M g C I 2 • 6 H 2 0 and 247 g M g S O 4 • 7H2O per liter of purest available water and sterilize by filtration through a prerinsed 0.2-/~m filter unit. Similarly prepare a 2 M stock of glucose (360 g/liter with purest water), sterilize by filtration, and store frozen in aliquots. For SOB and SOC, combine the magnesium and glucose as specified with autoclaved SOB-Mg medium, then sterilize by filtration through a 0.2-/~m filter unit just prior to use. It is advisable to use detergentfree filter units for all of these purposes. The final pH of the media should be 6.8 to 7.2. TB Medium for Plasmid Preparations and Stabilizing Unstable Inserts. TB medium4 has proved to be beneficial for maximizing the yield ofplasmid DNA from E. coli transformed with plasmids based on the high copy number pUC vectors. In addition, this medium facilitates the stabilization 4 R. Tartof and C. Hobbs, BRL Focus 9, 2 (1987).
[4]
71
PLASMID TRANSFORMATION
of inserts that are unstable and prone to rearrangement, as discussed in Section VI,B,4. PLASMID PREPARATION MEDIUM TB Compound
Amount/liter
Final concentration
Solution a Bacto-tryptone 12 g 1.2% Bacto yeast extract 24 g 2.4% Redistilled glycerol 4g 0.4% Combine in 900 ml water and autoclave Solution b KH2PO4 2.3 g 17 mM K2HPO 4 12.5 g 72 mM Dissolve the two potassium phosphates in 100 ml water and autoclave When solutions (a) and (b) are cool, combine them (1 : 1) to give complete TB medium
III. Protocols for Preparing Competent Escherichia coli Using Chemical Treatments The original discovery by Mandel and Higa ~that incubation of E. coli at low temperatures in the presence of calcium chloride induced a state of competence for DNA uptake (phase transfection) has in subsequent years been extended to include a variety of different chemicals and protocols for inducing competence for stable plasmid transformation. There have proved to be two major alternative chemical treatment regimens for inducing high degrees of competence. Each seems to be favored by different strains of E. coil; it is likely that genetic differences in the cell envelope determine susceptibility to transformation by one or the other of these conditions (J. Jessee and F. Bloom, unpublished results). The first method extends the simple Ca 2÷-based procedure through the utilization of manganese and potassium in addition to calcium. The second method originated from a series of experiments into the mechanisms of transformation, conducted primarily with an E. coli strain called DH1.5 This protocol produced very high transformation efficiencies using DH1 and many other strains. It is considerably more complex in its conditions, in that DMSO, DTT, and hexamine cobalt trichloride are employed in addition to those used in the simple Mn2+/Ca2+-based version. Each method is suitable for either immediate transformation or for frozen storage of competent cells, and 5 D.Hanahan, J. Mol. Biol. 166, 557 (1983).
72
Escherichia coli ANt) Salmonella typhimurium
[4]
variations for each purpose are described below. In addition, two convenient, rapid transformation protocols for routine use are described. The application of the various protocols for different strains and purposes is then considered. One significant new development in the methodology for preparing frozen competent cells is the observation that growth of the cells at lower temperatures than 37 ° can improve competence following a freeze-thaw cycle. 5a'Sb We have found that growth of E. coli at a temperature of 30° is preferable to 37° for most of the strains described below, using either protocols 5 or 7. 5a,5cFollowing the submission of this review, Inoue et al. 5b reported that growth at 18° is optimal for a new procedure which they have established (at press time we have not been able to evaluate this method). Thus, cell growth at lower temperatures than 37° can be added to the incubation in transformation buffers at 0° and the 42 ° heat pulse (or heat shock) as significant conditions for the induction of competence for transformation. A. M o d e r a t e Efficiency M e t h o d s B a s e d on M n 2+/Ca 2 + Treatment
MC10616 and its derivative, DH10B, 7 are preferentially transformed by a modification of the Mandel and Higa method. 1 These strains differ significantly from those derived from Hoffman Berling strain 11008 (e.g., MM294, DH1, DH5, DH5a, DH5aMCR) as well as from many other strains (e.g., HB101, C600) in that Mg 2+ is not beneficial in the growth medium, and the addition of either DMSO or DTT to the transformation buffer reduces competence. The following protocol works very well for MC1061 derivatives and may be preferable for certain other E. coli strains as well.
Protocol 5: Calcium/Manganese-Based (CCMB) Transformation for MC1061 Derived Strains 1. Pick several 2-3 mm diameter colonies from a freshly streaked plate into 1 ml of SOB-Mg growth medium (SOB without Mg). Avoid collecting agar fragments. Vortex gently to disperse cells. 5a j. Jessee and F. Bloom, U.S. Patent #4,981,797 (1991). 5b H. Inoue, H. Nojima, and H. Okayama, Gene 96, 23 (1990). J. Jessee and F. Bloom, unpublished observations. 6 M. Casadaban and S. Cohen, J. Mol. Biol. 138, 179 (1980). 7 S. G. N. Grant, J. Jessee, F, R. Bloom, and D. Hanahan, Proc. Natl. Acad. Sci. U.S.A. 87, 4654 (1990). 8 M. Meselson and R. Yuan, Nature (London) 217, 1110 (1968).
[4]
PLASMID TRANSFORMATION
73
2. Inoculate the dispersed colonies into a shake flask containing SOB-Mg medium (50 ml medium in a 500-ml nonbaffled Erlenmeyer flask, or 200 ml in a 2.8-liter Fernbach flask). 3. Incubate at 275 rpm, 30° until the OD550 reaches 0.3, which corresponds to 5 × 107 cells/ml for MCI061. 4. Collect the cell suspension into sterile 50-ml polypropylene centrifuge tubes and chill on ice for 10 rain. (Take a 10-/zl aliquot to determine the density of viable cells by plating a 106 dilution onto a SOB agar plate.) 5. Pellet the cells at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 10-15 min at 4°. Decant the supernatant and invert the tubes to remove excess culture medium. 6. Disperse the ceils in 1/3 volume of CCMB 80 (see below) by gentle vortexing or rapping of the centrifuge tube. 7. Incubate on ice for 20 min. 8. Centrifuge for 10 min at 750-1000 g at 4 °. Decant as in Step 5. 9. Resuspend the cells in CCMB 80 at 1/12 of original volume. 10. The competent cells can be used immediately or aliquoted in 1-ml volumes into chilled 2-ml screw-capped polypropylene tubes, flash frozen in a dry ice/ethanol bath, and then stored at - 8 0 °.
For immediate transformation 11. Aliquot 200-p.1 volumes of the competent cell suspension into chilled Falcon 2059 polypropylene tubes. Incubate on ice for 10 min. 12. Add DNA in less than 20/.d of 0.5 x TE buffer. Incubate on ice for 30 min (0.5 x TE is 5 mM Tris, 0.2 mM EDTA, pH 7.4). 13. Heat-shock in a water bath at 42 ° for 90 sec. Place on ice for 2 min. 14. Add 800/zl of SOC medium. Incubate at 37 ° with mild agitation for 60 min. Then plate onto SOB agar plates with appropriate drug selection and incubate at 37° to develop colonies of the transformed cells. For frozen competent cells 10a. Remove frozen competent cells from the freezer. Thaw on ice. Then follow steps 11-14 above.
Escherichia coli AND Salmonella typhimurium
74
[4]
Materials TRANSFORMATION BUFFER CCMB 80
Compound
Amount/liter
Final concentration
CaC12 • 2H20 MnC12 • 4H20 MgCI2 • 6H20 Potassium acetate Redistilled glycerol
11.8 g 4.0 g 2.0 g 10 ml of a 1 M stock (pH 7.0) 100 ml
80 mM 20 mM 10 mM 10 mM 10% (v/v)
Preparation. First prepare a 1 M solution of potassium acetate and adjust to pH 7.0 using KOH, then sterilize by filtration through a prerinsed 0.2-/zm membrane and store frozen. Then prepare a solution of 10 mM potassium acetate, 10% glycerol (v/v) using these reagents and the purest available water. Add salts as solids and allow each to enter into solution before adding the next. Adjust pH to 6.4 with 0.1 N HCI. Do not adjust pH upward with base. Sterilize the solution by filtration through a prerinsed 0.22-/z filter and store at 4°. The formulation of SOB-Mg is given in Section II,C,2 above. B. High-Efficiency Transformation Methods Utilizing Complex Conditions (TFB/FSB) The high-efficiency transformation procedures described below are effective with many E. coli strains, including several which have wide applicability in molecular cloning experiments (DH5, DH5a, HB 101). The method and its evaluation has been described in considerable depth previously, a,5 Moreover, detailed descriptions of the evaluation and optimization of this method, as well as troubleshooting strategies, important parameters, and variations (e.g., for the E. coli strain X1776), can be found in these papers. The critical parameters for success with this method are as follows: (1) growth of the cells in medium containing magnesium (20 mM); (2) collection of the cells during mid log-phase growth following an "appropriate" inoculation regimen; (3) use of ultrapure water lacking organic contaminants; and (4) use of DMSO free of significant oxidation. When this method is used with these parameters satisified, very high transformation frequencies can routinely be achieved (2-3% of the plasmid molecules affecting a transformed cell; 5-10% of the cells rendered competent for transformation). On average, the immediate transformation procedure (Protocol 6) gives higher frequencies than does the frozen competent version (Protocol 7, which of necessity omits DTT). Interestingly, some commercial preparations of competent cells often equal or exceed those
[4]
PLASMID TRANSFORMATION
75
obtained with these protocols, and they can be considered as a valid option to preparing high-efficiency competent cells in the laboratory. Protocol 6. TFB-Based Chemical Transformation Protocol
1. Pick several 2-3 mm diameter colonies from a freshly streaked SOB agar plate and disperse in I ml of SOB medium by vortexing. Use one colony per 10 ml of culture medium. The cells are best streaked from a frozen stock or fresh stab about 16-20 hr prior to initiating liquid growth. 2. Inoculate the cells into an Erlenmeyer flask containing SOB medium. Use a culture volume to flask volume ratio between 1 : 10 and 1 : 30 (e.g., 30-100 ml in a l-liter flask). 3. Incubate at 37 ° with moderate agitation until the cell density is 4-7 × 10 7 viable ceUs/ml (OD550 0.4 for DH5, 0.5 for DH5a and DH5aF'). 4. Collect the culture into 50-ml polypropylene centrifuge tubes (such as Falcon 2070 tubes) and chill on ice for 10-15 min. (Take a 10/zl aliquot of cells to determine the density of viable cells by plating a 106 dilution onto an SOB/agar plate.) 5. Pellet the cells by centrifugation at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 12-15 min at 4°. Drain the pelleted cells thoroughly, by inverting the tubes on paper towels and rapping sharply to remove any liquid. A micropipette can be used to draw off recalcitrant drops. 6. Resuspend the cells in 1/3 of the culture volume of TFB (see below) by vortexing moderately. Incubate on ice for 10-15 min. 7. Pellet the cells and drain thoroughly as in Step 5. 8. Resuspend the cells in TFB to 1/12.5 of the original volume. This represents a concentration from each 2.5 ml of culture into 200 txl of TFB. 9. Add DMSO and DTT solution (DnD, see below) to 3.5% (v/v) (7 t~l per 200/zl of cell suspension). Squirt the DnD into the center of the cell suspension and immediately swirl the tube for several seconds. Incubate the tubes on ice for 10 min. 10. Add a second, equal aliquot of DnD as in Step 9 to give a 7% final concentration. Incubate the tubes on ice for 10-20 min. 11. Pipette 210-/zl aliquots into chilled 17 x 100 mm polypropylene tubes (Falcon 2059 or equivalent). 12. Add the DNA solution in a volume of less than 20/zl, swirling to mix. Incubate the tubes on ice for 20-40 min. (Ligations should be diluted or precipitated in ethanol.)
76
Escherichia coli AND Salmonella typhimurium
[4]
13. Heat-shock the cells by placing the tubes in a 42 ° water bath for 90 sec. Return the tubes to ice to quench the heat shock, allowing 2 min for cooling. 14. Add 800/zl of SOC medium to each tube. Incubate at 37 ° with moderate agitation for 30-60 min. Spread the cells on agar plates containing appropriate antibiotics (or other conditions) to select for transformants. The incubation period should be omitted for M13 transfections. Materials STANDARD TRANSFORMATION BUFFER (TFB) Compound
Amount/liter
Final concentration
KCI (ultrapure) MnC1 • 4H20 CaCl2 • 2H20 HACoCI 3 Potassium MES (Final pH 6,20 -+ 0.10)
7.4 g 8.9 g 1.5 g 0.8 g 20 ml of 0,5 M stock (pH 6.3)
100 mM 45 mM 10 mM 3 mM 10 mM
Preparation. Equilibrate a 0.5 M solution of MES [2(N-morpholino) ethane sulfonic acid] to pH 6.3 using concentrated KOH, then sterilize by filtration through a 0.2-/~m membrane and store in aliquots at - 2 0 °. Make a solution of 10 mM potassium MES, using the 0.5 M MES stock and the purest available water. Add the salts as solids, then filter the solution through a 0.22-/zm prerinsed membrane. HACoCI3 is hexamminecobalt trichloride. Aliquot into sterile flasks and store at 4°. (TFB is stable for over 1 year.) DMSO AND DTT SOLUTION (DnD) Compound
Amount/10 ml final volume
Final concentration
DTT DMSO (spectroscopy grade) Potassium acetate
1.53 g 9 rrd 100/.d of a 1 M stock (pH 7.5)
1M 90% (v/v) 10 mM
The formulation of SOB medium is given in Section II,C,2.
Protocol 7. FSB-Based Frozen Storage of Competent Cells 1. Pick several 2-3 mm diameter colonies from a freshly streaked SOB agar plate and disperse in 1 ml of SOB medium by vortexing.
[4]
PLASMID TRANSFORMATION
2.
3.
4.
5.
6.
7. 8. 9.
10. 11. 12.
13.
77
Use one colony per 10 ml of culture medium. The cells are best streaked from a frozen stock or fresh stab about 16-20 hr prior to initiating liquid growth. Inoculate the cells into an Erlenmeyer flask containing SOB medium. Use a culture volume to flask volume ratio between 1 : 10 and 1 : 30 (e.g., 30-100 ml in a l-liter flask). Incubate at 30° with moderate agitation until the cell density is 6-9 × 10 7 viable cells/ml (OD550 0.4 for DH5, 0.5 for DH5et and DH5aF'). Collect the culture into 50-ml polypropylene centrifuge tubes (Falcon 2070 tubes) and chill on ice for 10-15 min. (Take an aliquot of cells to determine viable cell density by plating a 106 dilution onto an SOB agar plate.) Pellet the cells by centrifugation at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 12-15 min at 4 °. Drain the pelleted cells thoroughly, by inverting the tubes on paper towels and rapping sharply to remove any liquid. A micropipette can be used to draw off recalcitrant drops. Resuspend the cells in I/3 of the culture volume of frozen storage buffer (FSB, see below) by vortexing moderately. Incubate on ice for 10-15 min. Pellet the cells as before and drain thoroughly as in Step 5. Re suspend the cells in FSB to 1/12.5 of the original volume. (Each 2.5 ml of culture is concentrated into 200/zl of FSB.) Add DMSO to 3.5% (v/v) (7 /~1 per 200/zl of cell suspension). Squirt the DMSO into the center of the cell suspension and immediately swirl the tube for several seconds. Incubate the tube on ice for 5 min. (DTT is not used in this protocol.) Add a second, equal aliquot of DMSO, as before, giving a 7% final concentration. Incubate on ice for 10-15 min. Pipette 210-/~1 aliquots into chilled screw-cap polypropylene tubes, 1.5-ml microcentrifuge tubes, or snap-cap polypropylene tubes. Flash freeze by placing the tubes in a dry ice/ethanol bath or into liquid nitrogen for several minutes. (Be careful that the ethanol does not get inside the tubes. An option is to avoid total immersion but instead just set the bottom half of the tubes into the bath.) Transfer tubes to a - 8 0 ° freezer.
Use of frozen competent cells 1. Remove a tube(s) from the freezer and thaw on ice. If small vials were used, it is recommended that the cells be transferred in 200/zl aliquots to 12-ml polypropylene tubes (e.g., Falcon 2059).
Escherichia coli AND Salmonella typhimurium
78
[4]
2. Add the DNA solution in a volume of less than 20/zl. Swirl the tube to mix the DNA evenly with the cells. 3. Incubate the tube(s) on ice for 10-30 min. 4. Heat-shock the cells by placing the tubes in a 42 ° water bath for 90 sec, and then chill by returning the tubes immediately to 0° (crushed ice). 5. Add 800/~1 of SOC medium and incubate at 37 ° with moderate agitation for 30-60 min.
Materials FROZEN STORAGE BUFFER (FSB) Compound
Amount/liter
Final concentration
KCI MnCI2 • 4H20 CaC12 • 2H20 HACoC13 Potassium acetate RedistiUed glycerol (Final pH 6.20 -+ 0.10)
7.4 g 8.9 g 1.5 g 0.8 g l0 ml of a 1 M stock (pH 7.5) 100 g
100 mM 45 mM 10 mM 3 mM 10 mM 10% (w/v)
Preparation. Equilibrate a I M solution of potassium acetate to pH 7.5 using KOH, then sterilize by filtration through a 0.2-/zm membrane and store frozen. Prepare a 10 mM potassium acetate, 10% glycerol solution using this stock and the purest available water. Add the salts as solids, and adjust the pH (if necessary) to 6.4 using 0.1 N HCI. Do not adjust the pH upward with base. (The pH may drift for 1-2 days before settling at 6.1-6.2.) Sterilize the solution by filtration through a prerinsed 0.2-/zm filter and store at 4 °. HACoC13 is hexamminecobalt trichloride. SOB and DMSO are described in Section II,C,2. C. PEG/DMSO-Mediated Transformation Competence induction in E. coli by treatment with polyethylene glycol (PEG) and DMSO is a convenient procedure which produces moderate transformation frequencies. 9 This method may also be useful for transformation of other gram-negative microorganisms. Transformation efficiencies of 5 x 106 to 108 transformants//zg can be obtained with many E. coli strains. No heat shock is required for this procedure. 9 C. Chung, S. Neimela, and R. Miller, Proc. Natl. Acad. Sci. U.S.A. 86, 2172 (1989).
[4]
PLASMID TRANSFORMATION
79
Protocol 8. PEG/DMSO One-Step Transformation Procedure 1. I n c u b a t e E. coli at 37 ° in L B b r o t h to OD550 0 . 4 - 0 . 5 , c o r r e s p o n d i n g to a cell d e n s i t y o f 5 × l07 ceUs/ml. 2. Pellet the cells b y c e n t r i f u g a t i o n at 1000 g for 10 min at 4 °. R e s u s p e n d in 1/10 v o l u m e cold t r a n s f o r m a t i o n and storage solution (TSS, see below). 3. I n c u b a t e the cells o n ice for 20 min. 4. T r a n s f e r 100-/~1 aliquots o f the cell s u s p e n s i o n into chilled p o l y p r o p y l e n e t u b e s and mix with 100 p g - 1 ng D N A . I n c u b a t e on ice f o r 30 min. ( A n y remaining cells c a n be f r o z e n in d r y i c e / e t h a n o l and s t o r e d at - 8 0 ° f o r s u b s e q u e n t use.) 5. A d d 0.9 ml L B m e d i u m c o n t a i n i n g 20 m M g l u c o s e (or use S O C ) and i n c u b a t e at 37 ° with m o d e r a t e agitation (225 rpm) f o r 1 hr. 6. Plate cells, diluting into L B or S O C if n e c e s s a r y .
Materials TSS COMPONENTS Compound a. LB broth Bacto-tryptone Bacto yeast extract NaCI Dissolve in 1 liter ultrapure water and autoclave b. 2.0 M Mg2+ solution 1 M MgC1z . 6H:O 1 M MgSO4 • 7H20 Dissolve in 1 liter ultrapure water and filter sterilize c. 2 M glucose solution Prepare in 1 liter ultrapure water and sterilize by filtration d. Polyethylene glycol (PEG) e. Dimethyl sulfoxide (DMSO)
Amount/liter
Final concentration in TSS
10 g 5g 5g
-0.85% -0.4% - 8 mM
203 g 247 g
lOmM lOmM
360 g
20 mM
Use either molecular weight 3350 or 8000; add as a solid directly into TSS Ultrapure spectroscopy grade
10% 5%
80
Escherichia coli AND Salmonella typhimurium
[4]
Preparation of Transformation and Storage Solution (TSS) 1. Add solid PEG to LB to make a 10% (w/v) solution. 2. Add an aliquot of the 2.0 M M g 2÷ solution to achieve a final concentration of 20 mM. 3. Measure the pH, which should be about 6.8. If the pH is higher, the solution can be titrated with 1.0 N HCI. (The final pH of the TSS should be between 6.5 and 6.8.) 4. Filter sterilize the solution through a standard 0.45-/xm filter unit. 5. Add DMSO to the filtered solution to a final concentration of 5% (v/v). Store TSS at 4 ° or on ice until ready to use. Alternatively, the PEG, Mg 2÷ , and DMSO can be added to the LB, the pH adjusted, and the solution sterile filtered through a 0.2-/zm nylon filter unit.
D. Rapid Colony Transformation It is possible to collect a few colonies of cells from an agar plate, disperse them in transformation buffer, chill on ice to induce competence, and add DNA to effect transformation. This procedure is extremely fast and easy. It is limited in efficiency, however, and typically gives transformation efficiencies (XFE values) of ltP//.~g pBR322. It is not recommended for primary cloning experiments but is very useful for reintroducing cloned plasmid DNA back into E. coli prior to growing up large-scale cultures for plasmid DNA purification and other purposes.
Protocol 9. Rapid Colony Transformation 1. Pick several colonies (or a clump of cells) from a plate using a tungsten inoculating loop or a wooden applicator stick, being careful to take no agar along with the cells. 2. Disperse the colonies in 200/~1 of chilled standard transformation buffer (TFB) by vigorous vortexing or by repeated pipetting (CCMB 80 or FSB can be used instead of TFB). 3. Incubate the cells on ice for 10 min. 4. Add DNA solution (10-1000 ng) in less than 20 p.l, swirl to mix, and incubate on ice for 10 min. 5. Heat-shock the cells at 37-42 ° for 90 sec. (This step is optional if >100 ng of DNA is used.) 6. Add 400-800/zl SOC medium and incubate 20-60 min at 37°. (This step is also optional and unnecessary if > I00 ng of DNA is used in the transformation.) 7. Plate several fractions (e.g., I%, 5%, 25%) on appropriate selective media. Incubate to establish colonies.
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PLASMID TRANSFORMATION
81
E. Choice of Transformation Method Bacterial transformations can be divided into three classes with regard to purpose and consequent requirements for transformation efficiency: (1) primary cloning of high-complexity plasmid populations, for example, cDNAs representing an mRNA population or fragments of the genome of an organism, (2) cloning of more restricted populations of plasmids, for example, following in vitro manipulations such as oligonucleotide mutagenesis, or subcloning fragments of previously cloned DNAs, and (3) reintroduction of a pure plasmid population (a single clone) for purposes such as the preparation of large quantities of plasmid DNA. The latter purpose does not require cells with high competence, and various methods will suffice. Among these are the rapid colony transformation (Protocol 9), the PEG/DMSO one-step procedure (Protocol 8), or methods employing frozen competent cells (Protocols 5 and 7). If frozen competent cells are used, we suggest older lots which have decayed in competence, since these are nevertheless more than adequate for the purpose. Alternatively, a small clump of highly competent frozen cells can be quickly scraped off the surface of a frozen aliquot, which is returned immediately to the freezer without thawing. A 5- to 10-/zl clump of high-competence cells is sufficient for retransforming a cloned plasmid into E. coli. The intermediate purpose of manipulating and modifying previously cloned DNA usually requires transformation efficiencies in the range of 10 7 to I08 transformants//zg. These values are achieved with the immediate and frozen storage versions of the high-efficiency transformation methods (Protocols 5-7), and to some extent with the PEG/DMSO one-step procedure (Protocol 8). Experience will to some extent dictate the choice of method. If insufficient numbers of transformants are being generated, the options are to increase either the number of discrete transformations performed or the amount of DNA being used, or to change the type of transformation procedure (to improve the competence). For some types of in vitro modifications, very high efficiencies may be necessary to obtain the desired number of transformants, in which case the highest efficiency methods should be used. Transformations which are intended to represent a high-complexity population or transformations with very small amounts of DNA (e.g., plasmid rescue from total genomic DNA) require very high efficiency transformation protocols. It is increasingly clear that the electroshock transformation method described below is the most efficient, and therefore it is the method of choice for such purposes. The method suffers from the requirement for a high-voltage electroporation device, and from the large volume of cells that must be grown during the preparation of electrocompe-
82
Escherichia coli AND Salmonella typhimurium
[4]
tent cells. The latter problem is being obviated by the availability of commercial preparations of concentrated cells for electroshock transformation. A protocol for electroshock transformation is given in Section IV,A (Protocol 10). An alternative to electroshock transformation is the use of the highefficiency chemical transformation procedures under conditions that produce optimal competence. Both Protocol 7 with DH5a and Protocol 5 with DH10B will produce transformation efficiencies in excess of 10 9 transformants//zg pBR322, provided that all the important parameters of the protocols are carefully satisfied. Given careful attention to the transformation protocols, it is routinely possible to achieve high-efficiency competent cell preparations. It is noted in addition that frozen competent cells in all four competence ranges are commercially available, including maximally efficient chemically competent cells and electrocompetent cells. This alternative may prove reasonable for investigators who prefer to avoid the rigors of achieving routine high-efficiency transformations. However, all of the procedures presented herein will provide reliable transformations, given commitment to the details of the protocols. F. Optimization o f Competence for New Escherichia coli Strains If the ultimate goal of a transformation experiment requires the use of a strain ofE. coli that has not been described here, nor in previous reports by Hanahan, 3'5 it may be necessary to determine which transformation protocol is most suited for the strain. It is first recommended that trial transformations be performed on the new strain using Protocols 5 (CCMB based) and 6 (TFB based). For the CCMB protocol cells should be grown without added magnesium (in SOB-Mg), whereas for the TFB-based version complete SOB medium is recommended. In addition, with the TFB version, the procedure can be tried without DMSO, without DTT, or without both. Both the density at which the cells are collected following their growth in SOB medium ( + / - Mg2÷) and the temperature at which the cells are grown can be varied. Regarding growth temperature, based on recent results one could consider testing new strains over a range from 18° to 37°. 5a'Sb If neither of these protocols gives adequate competence, the PEG/DMSO one-step procedure (Protocol 8) could be tried, as well as other variations on transformation buffers given by Hanahan. 3 Finally, electroshock transformation can be assessed,using the procedure given below (Protocol 10) or the variations suggested in Section V,C. It is our experience that most E. coli strains can be rendered competent to at least 10 7 transformants//xg, given a serious optimization process. However, unless the intended purpose specifically requires a specific new strain, the
14]
PLASMID TRANSFORMATION
83
strains recommended in Table II should be considered, as they have proved to be reliable hosts for plasmid tranformation.
IV. Induction o f Competence with Electroshock Treatment The treatment of cells with a brief pulse of high-voltage electricity has been found to permeabilize them toward entry of a variety of macromolecules. 1°'1~ It is presumed that the discharge of a voltage potential across a field which includes cells transiently depolarizes their membranes and induces pores which can be entry points for macromolecules. Such electroshock treatment has recently been shown to be applicable to competence induction of E. coli and other bacteria. For E. coli, electroshock transformation is the most efficient method available and approaches the theoretical maximum of 100% cell transformation frequencies. Efficiencies of greater than 101° transformants/ttg of pUC18/19 have been reported, 12,~3 and greater than 80% of all cells can be transformed by this method. More recent improvements in electronics, E. coli strains, and procedures have increased the transformation efficiency up to 5 × 10~°, as is described below) 4 For other bacteria, electroporation is the only method presently available for the efficient introduction of plasmid DNA. The exposure of a dense suspension of bacterial cells and plasmid DNA to a high-strength electric field of short duration has been found to induce DNA uptake and to elicit competence for stable plasmid transformation. The generation of an electroshock is usually accomplished by the discharge of a high-voltage capacitor through a mixture of bacterial cells and DNA suspended between two electrodes. The pulse length of the capacitor discharge can be varied by increasing the capacitor size or the resistance in the circuit itself, which includes the mixture of cell suspension and DNA. A parallel resistor can also be used to modulate the resistance of the electroshock circuit. The time of the electric current pulse (the shock) is described by a decay time constant ~', which corresponds to the time at which the voltage has dropped to approximately 37% of its original value. The time constant of the electroshock is determined by the product 0" = RC) of the resistance R (both of the cell/DNA mixture and any parallel resistor) and the capacitance C of the circuit through which the electric field is being discharged. 10 D. Knight, in "Techniques in Cellular Physiology" (P. F. Baker, ed.), pp. 1-20. Elsevier, Amsterdam, 1981. ii E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. Hofschneider, EMBO J. 7, 841 (1982). 12 W. Dower, W. Miller, and C. Ragsdale, Nucleic Acids Res. 16, 6127 (1988). 13 W. Calvin and P. Hanawalt, J. Bacteriol. 170, 2796 (1988). 14 M. Smith, J. Jessee, T. Landers, and J. Jordan, Focus 12, 38 (1990).
84
[4]
Escherichia coli AND Salmonella typhimurium
X X X X X X X X
XXXX~'
m
.0.>
X
X
X
X
X
X
X
X
X X X X~
m
' ~ ' ~ 5 , ~ ' ~ , ~ , ~ -,- , 0
z 0 < Z
i l t l
+ + 1 1 + ÷ + 1
I,,,l
o z
=i +÷++
I 1 ÷ + 1 1 1 +
+++1
I 1 ÷ + ÷ + ÷ +
Z
+~ =.~
PLASMID TRANSFORMATION
63
[4] P l a s m i d T r a n s f o r m a t i o n o f E s c h e r i c h i a coli and Other Bacteria B y DOUGLAS HANAHAN, JOEL JESSEE,
and FREDRIC R. BLOOM
I. Introduction The discoveries that phage 1 and plasmid 2 DNAs could be transferred directly into bacterial cells following a competence induction process involving treatment with calcium chloride at low temperatures set the stage for molecular cloning of recombinant DNAs. The technique of DNA transformation has become important in virtually all aspects of molecular genetics. Escherichia coli has developed into a universal host organism both for molecular cloning of DNA and for a diverse set of assays involving clones genes. Transformation of other bacteria is also being applied for more specialized purposes, in addition to genetic studies of these organisms themselves, as the chapters throughout this volume testify. This chapter presents the major techniques and parameters that affect transformation of bacteria. The principal focus is on E. coli, although the basic principles for effectively transforming other gram-negative and grampositive bacteria are discussed as well. There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. There are two primary technical variations: chemical induction of competence and highvoltage electroshock treatment (electroporation). Both the characteristic of the cells being transformed and the purpose of the transformation will affect the choice of method. The second major parameter is the genetic constitution of the host strain of the organism being transformed, as there is clear evidence that a variety of genes can dramatically influence the outcome of transformation experiments. Since these genetic factors are increasingly well understood, they are discussed in some depth below. Methods for inducing competence by chemical treatments and electroshock are described, along with other relevant parameters, such as handling of bacteria for transformation purposes. Finally, application of these principles (especially electroshock treatment) to other bacteria is discussed. The E. coli transformation protocols to be presented are summarized in Table I. I M . Mandel and A. Higa, J. Mol. Biol. 53, 159 (1970). 2 S. Cohen, A. Chang, and L. Hsu, Proc. Natl. Acad. Sci. U.S.A. 69, 2110 (1972).
METHODS IN ENZYMOLOGY, VOL. 204
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
64
Escherichia coli AND Salmonella typhimurium
[4]
TABLE I
Escherichia coli TRANSFORMATION METHODSa Typical Typical transformation competence efficiency (F0 (XFE) (%)
Protocol
Method
5
Calcium manganesebased (CCMB)
1-10 x 10s
1-5
MC1061, DH10B
6
TFB-based high efficiency
0.4-2 x 109
2-10
DHI, DH5, HB 101
7
FSB-based frozen competent
1-4
DH1, DH5, HBI01
8
PEG/DMSO one step
106-107
0.01-0.1
Most common cloning hosts
9
Rapid colony transformation
103-104
90%). 2b. For long-term storage, resuspend the cell pellet in a mixture of 80% SOB medium/20% redistilled glycerol, transfer the cell suspension to a 1.5 to 2-ml polypropylene tube (preferably screw-capped), chill on ice, flash freeze in a dry ice/ethanol bath, and place at - 80°. C. Reagents and Growth Media Both chemical and electroshock methods of transformation show some sensitivity to the quality of reagents used in the transformation buffers. This is particularly true for the high-efficiency protocols which employ dimethyl sulfoxide (DMSO), where it appears that organic contaminants, including oxidation products of DMSO itself, are highly inhibitory to the induction of competence for plasmid transformation. With regard to growth medium, there has also been variability noted in lots of digested casein (tryptone) and e~tracts of yeast with regard to competence induction. Besides possible effects of reagent quality, the transformation methods differ in the benefits of elevated levels (20 mM) of magnesium during cell growth prior to competence induction. The protocols using standard transformation buffer (TFB) or frozen storage buffer (FSB) and DMSO benefit from added magnesium, whereas the chemical protocol optimized for E. coli MC 1061 derivatives and the electrocompetenceprotocol require growth without added magnesium. 1. Quality and Suppliers. As a general rule ultrapure or reagent-grade chemicals should be employed, and most major suppliers are satisfactory (including Fluka, Ronkonkoma, NY; Mallinkrodt, St. Louis, MO; and Fisher, Fairlawn, N J). The quality of the water is very important for the procedures using DMSO and can impact on all of the methods. In general we recommend the use of water purified by reverse osmosis, as this procedure effectively removes not only salts but also organic contaminants, including those with similar volatility to water, which are not re-
[4]
PLASMID TRANSFORMATION
69
moved by distillation. However, it is important that the carbon filters which remove organics be replaced regularly, as there is no gauge to measure their saturation (except drastically reduced competence when it occurs). Distilled or deionized water can also be further purified in the laboratory with carbon, as described by Hanahan. 3 Dimethyl sulfoxide (DMSO) is subject to oxidation, and its oxidation products can be a major source of variability in the levels of competence induced by procedures which employ it. Note also that DMSO dissolves polystyrene, and therefore tubes composed of this material cannot be used. We recommend that DMSO be purchased in the smallest possible amounts (e.g., 100-ml bottles) at spectroscopy grade, aliquoted to fill small (0.5 ml) polypropylene tubes completely, and then stored at - 80°. A tube of DMSO is thawed, used for that day, and then discarded. Among the adequate suppliers of DMSO are Fluka, Mallinkrodt, and MCB. Similarly, the dithiothreitol (DTT) used in the TFB method (Protocol 6) is also subject to impurities which inhibit competence. One of the most reliable suppliers of DTT is Calbiochem (La Jolla, CA). Impurities in glycerol products can affect all of the competence and storage protocols used here, and it is recommended that redistilled or spectroscopy grades of glycerol be used. Among the recommended suppliers are Fluka and BRL (Gaithersburg, MD). Other variables which can influence competence induction are in the flasks and tubes used for culturing and treating the cells during transformation. Of particular concern are soap residues in glassware and surfactants used in manufacturing some brands of polypropylene tubes. It is recommended that glassware used in transformation procedures be thoroughly rinsed with distilled water, and autoclaved half full of water, to prevent deposition of surface residues (including soap). For E. coli we do not use glassware washing services but rather wash by hand without soap. Among the reliable suppliers of polypropylene tubes for transformation procedures are Falcon (Oxnard, CA) and Coming (Coming, NY). The components of the growth media can also influence transformation. We routinely use Bacto-tryptone and Bacto yeast extract. Alternative suppliers should be carefully compared, as lot variations have been reported with regard to transformation experiments. Again, reagent-grade chemicals and the purest available water are recommended. 2. Formulations of Media Used for Cell Growth and Recooery in Transformation Procedures. All the transformation protocols described herein use a common set of growth media, with the only major distinction 3 D. Hanahan, in " D N A Cloning Techniques" (D. Glover, ed.), p. 109. IRL Press, London, 1985.
70
Escherichia coli AND Salmonella typhimurium
[4]
being the presence or absence of elevated levels of magnesium. These differ from many traditional media in that the levels of sodium are low. The recovery of cells from all of these procedures is enhanced in SOC medium, which contains glucose. We recommend that these media be filter sterilized just prior to use, to remove particulate matter and any slow-growing contaminating microorganisms. Each medium is identical to or derived from a stock of SOB-Mg medium (SOB without magnesium), and their formulations are as follows. Compound Growth medium SOB-Mg Bacto-tryptone Bacto yeast extract NaCl KCI Growth medium SOB SOB-Mg medium MgC12 and MgSO4 Recovery medium SOC S O B - M g medium MgC12 and MgSO4 Glucose
Amount/liter
Final concentration
20 g 5g 0.58 g 0.19 g
2.0% 0.5% 10.0 mM 2.5 mM
1 liter 10 ml of a 2 M stock
99% 20 mM
1 liter 10 ml of a 2 M stock 10 ml of a 2 M stock
98% 20 raM 20 mM
Preparation. Combine tryptone, yeast extract, NaC1, and KCI in the purest available water. Aliquot into prerinsed glass flasks and autoclave for 30-40 min to generate SOB-Mg medium. Sterilize by filtration just prior to use for protocols requiring this medium itself. Prepare a 2 M stock solution of M g 2+ by combining 203 g M g C I 2 • 6 H 2 0 and 247 g M g S O 4 • 7H2O per liter of purest available water and sterilize by filtration through a prerinsed 0.2-/~m filter unit. Similarly prepare a 2 M stock of glucose (360 g/liter with purest water), sterilize by filtration, and store frozen in aliquots. For SOB and SOC, combine the magnesium and glucose as specified with autoclaved SOB-Mg medium, then sterilize by filtration through a 0.2-/~m filter unit just prior to use. It is advisable to use detergentfree filter units for all of these purposes. The final pH of the media should be 6.8 to 7.2. TB Medium for Plasmid Preparations and Stabilizing Unstable Inserts. TB medium4 has proved to be beneficial for maximizing the yield ofplasmid DNA from E. coli transformed with plasmids based on the high copy number pUC vectors. In addition, this medium facilitates the stabilization 4 R. Tartof and C. Hobbs, BRL Focus 9, 2 (1987).
[4]
71
PLASMID TRANSFORMATION
of inserts that are unstable and prone to rearrangement, as discussed in Section VI,B,4. PLASMID PREPARATION MEDIUM TB Compound
Amount/liter
Final concentration
Solution a Bacto-tryptone 12 g 1.2% Bacto yeast extract 24 g 2.4% Redistilled glycerol 4g 0.4% Combine in 900 ml water and autoclave Solution b KH2PO4 2.3 g 17 mM K2HPO 4 12.5 g 72 mM Dissolve the two potassium phosphates in 100 ml water and autoclave When solutions (a) and (b) are cool, combine them (1 : 1) to give complete TB medium
III. Protocols for Preparing Competent Escherichia coli Using Chemical Treatments The original discovery by Mandel and Higa ~that incubation of E. coli at low temperatures in the presence of calcium chloride induced a state of competence for DNA uptake (phase transfection) has in subsequent years been extended to include a variety of different chemicals and protocols for inducing competence for stable plasmid transformation. There have proved to be two major alternative chemical treatment regimens for inducing high degrees of competence. Each seems to be favored by different strains of E. coil; it is likely that genetic differences in the cell envelope determine susceptibility to transformation by one or the other of these conditions (J. Jessee and F. Bloom, unpublished results). The first method extends the simple Ca 2÷-based procedure through the utilization of manganese and potassium in addition to calcium. The second method originated from a series of experiments into the mechanisms of transformation, conducted primarily with an E. coli strain called DH1.5 This protocol produced very high transformation efficiencies using DH1 and many other strains. It is considerably more complex in its conditions, in that DMSO, DTT, and hexamine cobalt trichloride are employed in addition to those used in the simple Mn2+/Ca2+-based version. Each method is suitable for either immediate transformation or for frozen storage of competent cells, and 5 D.Hanahan, J. Mol. Biol. 166, 557 (1983).
72
Escherichia coli ANt) Salmonella typhimurium
[4]
variations for each purpose are described below. In addition, two convenient, rapid transformation protocols for routine use are described. The application of the various protocols for different strains and purposes is then considered. One significant new development in the methodology for preparing frozen competent cells is the observation that growth of the cells at lower temperatures than 37 ° can improve competence following a freeze-thaw cycle. 5a'Sb We have found that growth of E. coli at a temperature of 30° is preferable to 37° for most of the strains described below, using either protocols 5 or 7. 5a,5cFollowing the submission of this review, Inoue et al. 5b reported that growth at 18° is optimal for a new procedure which they have established (at press time we have not been able to evaluate this method). Thus, cell growth at lower temperatures than 37° can be added to the incubation in transformation buffers at 0° and the 42 ° heat pulse (or heat shock) as significant conditions for the induction of competence for transformation. A. M o d e r a t e Efficiency M e t h o d s B a s e d on M n 2+/Ca 2 + Treatment
MC10616 and its derivative, DH10B, 7 are preferentially transformed by a modification of the Mandel and Higa method. 1 These strains differ significantly from those derived from Hoffman Berling strain 11008 (e.g., MM294, DH1, DH5, DH5a, DH5aMCR) as well as from many other strains (e.g., HB101, C600) in that Mg 2+ is not beneficial in the growth medium, and the addition of either DMSO or DTT to the transformation buffer reduces competence. The following protocol works very well for MC1061 derivatives and may be preferable for certain other E. coli strains as well.
Protocol 5: Calcium/Manganese-Based (CCMB) Transformation for MC1061 Derived Strains 1. Pick several 2-3 mm diameter colonies from a freshly streaked plate into 1 ml of SOB-Mg growth medium (SOB without Mg). Avoid collecting agar fragments. Vortex gently to disperse cells. 5a j. Jessee and F. Bloom, U.S. Patent #4,981,797 (1991). 5b H. Inoue, H. Nojima, and H. Okayama, Gene 96, 23 (1990). J. Jessee and F. Bloom, unpublished observations. 6 M. Casadaban and S. Cohen, J. Mol. Biol. 138, 179 (1980). 7 S. G. N. Grant, J. Jessee, F, R. Bloom, and D. Hanahan, Proc. Natl. Acad. Sci. U.S.A. 87, 4654 (1990). 8 M. Meselson and R. Yuan, Nature (London) 217, 1110 (1968).
[4]
PLASMID TRANSFORMATION
73
2. Inoculate the dispersed colonies into a shake flask containing SOB-Mg medium (50 ml medium in a 500-ml nonbaffled Erlenmeyer flask, or 200 ml in a 2.8-liter Fernbach flask). 3. Incubate at 275 rpm, 30° until the OD550 reaches 0.3, which corresponds to 5 × 107 cells/ml for MCI061. 4. Collect the cell suspension into sterile 50-ml polypropylene centrifuge tubes and chill on ice for 10 rain. (Take a 10-/zl aliquot to determine the density of viable cells by plating a 106 dilution onto a SOB agar plate.) 5. Pellet the cells at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 10-15 min at 4°. Decant the supernatant and invert the tubes to remove excess culture medium. 6. Disperse the ceils in 1/3 volume of CCMB 80 (see below) by gentle vortexing or rapping of the centrifuge tube. 7. Incubate on ice for 20 min. 8. Centrifuge for 10 min at 750-1000 g at 4 °. Decant as in Step 5. 9. Resuspend the cells in CCMB 80 at 1/12 of original volume. 10. The competent cells can be used immediately or aliquoted in 1-ml volumes into chilled 2-ml screw-capped polypropylene tubes, flash frozen in a dry ice/ethanol bath, and then stored at - 8 0 °.
For immediate transformation 11. Aliquot 200-p.1 volumes of the competent cell suspension into chilled Falcon 2059 polypropylene tubes. Incubate on ice for 10 min. 12. Add DNA in less than 20/.d of 0.5 x TE buffer. Incubate on ice for 30 min (0.5 x TE is 5 mM Tris, 0.2 mM EDTA, pH 7.4). 13. Heat-shock in a water bath at 42 ° for 90 sec. Place on ice for 2 min. 14. Add 800/zl of SOC medium. Incubate at 37 ° with mild agitation for 60 min. Then plate onto SOB agar plates with appropriate drug selection and incubate at 37° to develop colonies of the transformed cells. For frozen competent cells 10a. Remove frozen competent cells from the freezer. Thaw on ice. Then follow steps 11-14 above.
Escherichia coli AND Salmonella typhimurium
74
[4]
Materials TRANSFORMATION BUFFER CCMB 80
Compound
Amount/liter
Final concentration
CaC12 • 2H20 MnC12 • 4H20 MgCI2 • 6H20 Potassium acetate Redistilled glycerol
11.8 g 4.0 g 2.0 g 10 ml of a 1 M stock (pH 7.0) 100 ml
80 mM 20 mM 10 mM 10 mM 10% (v/v)
Preparation. First prepare a 1 M solution of potassium acetate and adjust to pH 7.0 using KOH, then sterilize by filtration through a prerinsed 0.2-/zm membrane and store frozen. Then prepare a solution of 10 mM potassium acetate, 10% glycerol (v/v) using these reagents and the purest available water. Add salts as solids and allow each to enter into solution before adding the next. Adjust pH to 6.4 with 0.1 N HCI. Do not adjust pH upward with base. Sterilize the solution by filtration through a prerinsed 0.22-/z filter and store at 4°. The formulation of SOB-Mg is given in Section II,C,2 above. B. High-Efficiency Transformation Methods Utilizing Complex Conditions (TFB/FSB) The high-efficiency transformation procedures described below are effective with many E. coli strains, including several which have wide applicability in molecular cloning experiments (DH5, DH5a, HB 101). The method and its evaluation has been described in considerable depth previously, a,5 Moreover, detailed descriptions of the evaluation and optimization of this method, as well as troubleshooting strategies, important parameters, and variations (e.g., for the E. coli strain X1776), can be found in these papers. The critical parameters for success with this method are as follows: (1) growth of the cells in medium containing magnesium (20 mM); (2) collection of the cells during mid log-phase growth following an "appropriate" inoculation regimen; (3) use of ultrapure water lacking organic contaminants; and (4) use of DMSO free of significant oxidation. When this method is used with these parameters satisified, very high transformation frequencies can routinely be achieved (2-3% of the plasmid molecules affecting a transformed cell; 5-10% of the cells rendered competent for transformation). On average, the immediate transformation procedure (Protocol 6) gives higher frequencies than does the frozen competent version (Protocol 7, which of necessity omits DTT). Interestingly, some commercial preparations of competent cells often equal or exceed those
[4]
PLASMID TRANSFORMATION
75
obtained with these protocols, and they can be considered as a valid option to preparing high-efficiency competent cells in the laboratory. Protocol 6. TFB-Based Chemical Transformation Protocol
1. Pick several 2-3 mm diameter colonies from a freshly streaked SOB agar plate and disperse in I ml of SOB medium by vortexing. Use one colony per 10 ml of culture medium. The cells are best streaked from a frozen stock or fresh stab about 16-20 hr prior to initiating liquid growth. 2. Inoculate the cells into an Erlenmeyer flask containing SOB medium. Use a culture volume to flask volume ratio between 1 : 10 and 1 : 30 (e.g., 30-100 ml in a l-liter flask). 3. Incubate at 37 ° with moderate agitation until the cell density is 4-7 × 10 7 viable ceUs/ml (OD550 0.4 for DH5, 0.5 for DH5a and DH5aF'). 4. Collect the culture into 50-ml polypropylene centrifuge tubes (such as Falcon 2070 tubes) and chill on ice for 10-15 min. (Take a 10/zl aliquot of cells to determine the density of viable cells by plating a 106 dilution onto an SOB/agar plate.) 5. Pellet the cells by centrifugation at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 12-15 min at 4°. Drain the pelleted cells thoroughly, by inverting the tubes on paper towels and rapping sharply to remove any liquid. A micropipette can be used to draw off recalcitrant drops. 6. Resuspend the cells in 1/3 of the culture volume of TFB (see below) by vortexing moderately. Incubate on ice for 10-15 min. 7. Pellet the cells and drain thoroughly as in Step 5. 8. Resuspend the cells in TFB to 1/12.5 of the original volume. This represents a concentration from each 2.5 ml of culture into 200 txl of TFB. 9. Add DMSO and DTT solution (DnD, see below) to 3.5% (v/v) (7 t~l per 200/zl of cell suspension). Squirt the DnD into the center of the cell suspension and immediately swirl the tube for several seconds. Incubate the tubes on ice for 10 min. 10. Add a second, equal aliquot of DnD as in Step 9 to give a 7% final concentration. Incubate the tubes on ice for 10-20 min. 11. Pipette 210-/zl aliquots into chilled 17 x 100 mm polypropylene tubes (Falcon 2059 or equivalent). 12. Add the DNA solution in a volume of less than 20/zl, swirling to mix. Incubate the tubes on ice for 20-40 min. (Ligations should be diluted or precipitated in ethanol.)
76
Escherichia coli AND Salmonella typhimurium
[4]
13. Heat-shock the cells by placing the tubes in a 42 ° water bath for 90 sec. Return the tubes to ice to quench the heat shock, allowing 2 min for cooling. 14. Add 800/zl of SOC medium to each tube. Incubate at 37 ° with moderate agitation for 30-60 min. Spread the cells on agar plates containing appropriate antibiotics (or other conditions) to select for transformants. The incubation period should be omitted for M13 transfections. Materials STANDARD TRANSFORMATION BUFFER (TFB) Compound
Amount/liter
Final concentration
KCI (ultrapure) MnC1 • 4H20 CaCl2 • 2H20 HACoCI 3 Potassium MES (Final pH 6,20 -+ 0.10)
7.4 g 8.9 g 1.5 g 0.8 g 20 ml of 0,5 M stock (pH 6.3)
100 mM 45 mM 10 mM 3 mM 10 mM
Preparation. Equilibrate a 0.5 M solution of MES [2(N-morpholino) ethane sulfonic acid] to pH 6.3 using concentrated KOH, then sterilize by filtration through a 0.2-/~m membrane and store in aliquots at - 2 0 °. Make a solution of 10 mM potassium MES, using the 0.5 M MES stock and the purest available water. Add the salts as solids, then filter the solution through a 0.22-/zm prerinsed membrane. HACoCI3 is hexamminecobalt trichloride. Aliquot into sterile flasks and store at 4°. (TFB is stable for over 1 year.) DMSO AND DTT SOLUTION (DnD) Compound
Amount/10 ml final volume
Final concentration
DTT DMSO (spectroscopy grade) Potassium acetate
1.53 g 9 rrd 100/.d of a 1 M stock (pH 7.5)
1M 90% (v/v) 10 mM
The formulation of SOB medium is given in Section II,C,2.
Protocol 7. FSB-Based Frozen Storage of Competent Cells 1. Pick several 2-3 mm diameter colonies from a freshly streaked SOB agar plate and disperse in 1 ml of SOB medium by vortexing.
[4]
PLASMID TRANSFORMATION
2.
3.
4.
5.
6.
7. 8. 9.
10. 11. 12.
13.
77
Use one colony per 10 ml of culture medium. The cells are best streaked from a frozen stock or fresh stab about 16-20 hr prior to initiating liquid growth. Inoculate the cells into an Erlenmeyer flask containing SOB medium. Use a culture volume to flask volume ratio between 1 : 10 and 1 : 30 (e.g., 30-100 ml in a l-liter flask). Incubate at 30° with moderate agitation until the cell density is 6-9 × 10 7 viable cells/ml (OD550 0.4 for DH5, 0.5 for DH5et and DH5aF'). Collect the culture into 50-ml polypropylene centrifuge tubes (Falcon 2070 tubes) and chill on ice for 10-15 min. (Take an aliquot of cells to determine viable cell density by plating a 106 dilution onto an SOB agar plate.) Pellet the cells by centrifugation at 750-1000 g (2000-3000 rpm in a clinical centrifuge) for 12-15 min at 4 °. Drain the pelleted cells thoroughly, by inverting the tubes on paper towels and rapping sharply to remove any liquid. A micropipette can be used to draw off recalcitrant drops. Resuspend the cells in I/3 of the culture volume of frozen storage buffer (FSB, see below) by vortexing moderately. Incubate on ice for 10-15 min. Pellet the cells as before and drain thoroughly as in Step 5. Re suspend the cells in FSB to 1/12.5 of the original volume. (Each 2.5 ml of culture is concentrated into 200/zl of FSB.) Add DMSO to 3.5% (v/v) (7 /~1 per 200/zl of cell suspension). Squirt the DMSO into the center of the cell suspension and immediately swirl the tube for several seconds. Incubate the tube on ice for 5 min. (DTT is not used in this protocol.) Add a second, equal aliquot of DMSO, as before, giving a 7% final concentration. Incubate on ice for 10-15 min. Pipette 210-/~1 aliquots into chilled screw-cap polypropylene tubes, 1.5-ml microcentrifuge tubes, or snap-cap polypropylene tubes. Flash freeze by placing the tubes in a dry ice/ethanol bath or into liquid nitrogen for several minutes. (Be careful that the ethanol does not get inside the tubes. An option is to avoid total immersion but instead just set the bottom half of the tubes into the bath.) Transfer tubes to a - 8 0 ° freezer.
Use of frozen competent cells 1. Remove a tube(s) from the freezer and thaw on ice. If small vials were used, it is recommended that the cells be transferred in 200/zl aliquots to 12-ml polypropylene tubes (e.g., Falcon 2059).
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2. Add the DNA solution in a volume of less than 20/zl. Swirl the tube to mix the DNA evenly with the cells. 3. Incubate the tube(s) on ice for 10-30 min. 4. Heat-shock the cells by placing the tubes in a 42 ° water bath for 90 sec, and then chill by returning the tubes immediately to 0° (crushed ice). 5. Add 800/~1 of SOC medium and incubate at 37 ° with moderate agitation for 30-60 min.
Materials FROZEN STORAGE BUFFER (FSB) Compound
Amount/liter
Final concentration
KCI MnCI2 • 4H20 CaC12 • 2H20 HACoC13 Potassium acetate RedistiUed glycerol (Final pH 6.20 -+ 0.10)
7.4 g 8.9 g 1.5 g 0.8 g l0 ml of a 1 M stock (pH 7.5) 100 g
100 mM 45 mM 10 mM 3 mM 10 mM 10% (w/v)
Preparation. Equilibrate a I M solution of potassium acetate to pH 7.5 using KOH, then sterilize by filtration through a 0.2-/zm membrane and store frozen. Prepare a 10 mM potassium acetate, 10% glycerol solution using this stock and the purest available water. Add the salts as solids, and adjust the pH (if necessary) to 6.4 using 0.1 N HCI. Do not adjust the pH upward with base. (The pH may drift for 1-2 days before settling at 6.1-6.2.) Sterilize the solution by filtration through a prerinsed 0.2-/zm filter and store at 4 °. HACoC13 is hexamminecobalt trichloride. SOB and DMSO are described in Section II,C,2. C. PEG/DMSO-Mediated Transformation Competence induction in E. coli by treatment with polyethylene glycol (PEG) and DMSO is a convenient procedure which produces moderate transformation frequencies. 9 This method may also be useful for transformation of other gram-negative microorganisms. Transformation efficiencies of 5 x 106 to 108 transformants//zg can be obtained with many E. coli strains. No heat shock is required for this procedure. 9 C. Chung, S. Neimela, and R. Miller, Proc. Natl. Acad. Sci. U.S.A. 86, 2172 (1989).
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79
Protocol 8. PEG/DMSO One-Step Transformation Procedure 1. I n c u b a t e E. coli at 37 ° in L B b r o t h to OD550 0 . 4 - 0 . 5 , c o r r e s p o n d i n g to a cell d e n s i t y o f 5 × l07 ceUs/ml. 2. Pellet the cells b y c e n t r i f u g a t i o n at 1000 g for 10 min at 4 °. R e s u s p e n d in 1/10 v o l u m e cold t r a n s f o r m a t i o n and storage solution (TSS, see below). 3. I n c u b a t e the cells o n ice for 20 min. 4. T r a n s f e r 100-/~1 aliquots o f the cell s u s p e n s i o n into chilled p o l y p r o p y l e n e t u b e s and mix with 100 p g - 1 ng D N A . I n c u b a t e on ice f o r 30 min. ( A n y remaining cells c a n be f r o z e n in d r y i c e / e t h a n o l and s t o r e d at - 8 0 ° f o r s u b s e q u e n t use.) 5. A d d 0.9 ml L B m e d i u m c o n t a i n i n g 20 m M g l u c o s e (or use S O C ) and i n c u b a t e at 37 ° with m o d e r a t e agitation (225 rpm) f o r 1 hr. 6. Plate cells, diluting into L B or S O C if n e c e s s a r y .
Materials TSS COMPONENTS Compound a. LB broth Bacto-tryptone Bacto yeast extract NaCI Dissolve in 1 liter ultrapure water and autoclave b. 2.0 M Mg2+ solution 1 M MgC1z . 6H:O 1 M MgSO4 • 7H20 Dissolve in 1 liter ultrapure water and filter sterilize c. 2 M glucose solution Prepare in 1 liter ultrapure water and sterilize by filtration d. Polyethylene glycol (PEG) e. Dimethyl sulfoxide (DMSO)
Amount/liter
Final concentration in TSS
10 g 5g 5g
-0.85% -0.4% - 8 mM
203 g 247 g
lOmM lOmM
360 g
20 mM
Use either molecular weight 3350 or 8000; add as a solid directly into TSS Ultrapure spectroscopy grade
10% 5%
80
Escherichia coli AND Salmonella typhimurium
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Preparation of Transformation and Storage Solution (TSS) 1. Add solid PEG to LB to make a 10% (w/v) solution. 2. Add an aliquot of the 2.0 M M g 2÷ solution to achieve a final concentration of 20 mM. 3. Measure the pH, which should be about 6.8. If the pH is higher, the solution can be titrated with 1.0 N HCI. (The final pH of the TSS should be between 6.5 and 6.8.) 4. Filter sterilize the solution through a standard 0.45-/xm filter unit. 5. Add DMSO to the filtered solution to a final concentration of 5% (v/v). Store TSS at 4 ° or on ice until ready to use. Alternatively, the PEG, Mg 2÷ , and DMSO can be added to the LB, the pH adjusted, and the solution sterile filtered through a 0.2-/zm nylon filter unit.
D. Rapid Colony Transformation It is possible to collect a few colonies of cells from an agar plate, disperse them in transformation buffer, chill on ice to induce competence, and add DNA to effect transformation. This procedure is extremely fast and easy. It is limited in efficiency, however, and typically gives transformation efficiencies (XFE values) of ltP//.~g pBR322. It is not recommended for primary cloning experiments but is very useful for reintroducing cloned plasmid DNA back into E. coli prior to growing up large-scale cultures for plasmid DNA purification and other purposes.
Protocol 9. Rapid Colony Transformation 1. Pick several colonies (or a clump of cells) from a plate using a tungsten inoculating loop or a wooden applicator stick, being careful to take no agar along with the cells. 2. Disperse the colonies in 200/~1 of chilled standard transformation buffer (TFB) by vigorous vortexing or by repeated pipetting (CCMB 80 or FSB can be used instead of TFB). 3. Incubate the cells on ice for 10 min. 4. Add DNA solution (10-1000 ng) in less than 20 p.l, swirl to mix, and incubate on ice for 10 min. 5. Heat-shock the cells at 37-42 ° for 90 sec. (This step is optional if >100 ng of DNA is used.) 6. Add 400-800/zl SOC medium and incubate 20-60 min at 37°. (This step is also optional and unnecessary if > I00 ng of DNA is used in the transformation.) 7. Plate several fractions (e.g., I%, 5%, 25%) on appropriate selective media. Incubate to establish colonies.
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PLASMID TRANSFORMATION
81
E. Choice of Transformation Method Bacterial transformations can be divided into three classes with regard to purpose and consequent requirements for transformation efficiency: (1) primary cloning of high-complexity plasmid populations, for example, cDNAs representing an mRNA population or fragments of the genome of an organism, (2) cloning of more restricted populations of plasmids, for example, following in vitro manipulations such as oligonucleotide mutagenesis, or subcloning fragments of previously cloned DNAs, and (3) reintroduction of a pure plasmid population (a single clone) for purposes such as the preparation of large quantities of plasmid DNA. The latter purpose does not require cells with high competence, and various methods will suffice. Among these are the rapid colony transformation (Protocol 9), the PEG/DMSO one-step procedure (Protocol 8), or methods employing frozen competent cells (Protocols 5 and 7). If frozen competent cells are used, we suggest older lots which have decayed in competence, since these are nevertheless more than adequate for the purpose. Alternatively, a small clump of highly competent frozen cells can be quickly scraped off the surface of a frozen aliquot, which is returned immediately to the freezer without thawing. A 5- to 10-/zl clump of high-competence cells is sufficient for retransforming a cloned plasmid into E. coli. The intermediate purpose of manipulating and modifying previously cloned DNA usually requires transformation efficiencies in the range of 10 7 to I08 transformants//zg. These values are achieved with the immediate and frozen storage versions of the high-efficiency transformation methods (Protocols 5-7), and to some extent with the PEG/DMSO one-step procedure (Protocol 8). Experience will to some extent dictate the choice of method. If insufficient numbers of transformants are being generated, the options are to increase either the number of discrete transformations performed or the amount of DNA being used, or to change the type of transformation procedure (to improve the competence). For some types of in vitro modifications, very high efficiencies may be necessary to obtain the desired number of transformants, in which case the highest efficiency methods should be used. Transformations which are intended to represent a high-complexity population or transformations with very small amounts of DNA (e.g., plasmid rescue from total genomic DNA) require very high efficiency transformation protocols. It is increasingly clear that the electroshock transformation method described below is the most efficient, and therefore it is the method of choice for such purposes. The method suffers from the requirement for a high-voltage electroporation device, and from the large volume of cells that must be grown during the preparation of electrocompe-
82
Escherichia coli AND Salmonella typhimurium
[4]
tent cells. The latter problem is being obviated by the availability of commercial preparations of concentrated cells for electroshock transformation. A protocol for electroshock transformation is given in Section IV,A (Protocol 10). An alternative to electroshock transformation is the use of the highefficiency chemical transformation procedures under conditions that produce optimal competence. Both Protocol 7 with DH5a and Protocol 5 with DH10B will produce transformation efficiencies in excess of 10 9 transformants//zg pBR322, provided that all the important parameters of the protocols are carefully satisfied. Given careful attention to the transformation protocols, it is routinely possible to achieve high-efficiency competent cell preparations. It is noted in addition that frozen competent cells in all four competence ranges are commercially available, including maximally efficient chemically competent cells and electrocompetent cells. This alternative may prove reasonable for investigators who prefer to avoid the rigors of achieving routine high-efficiency transformations. However, all of the procedures presented herein will provide reliable transformations, given commitment to the details of the protocols. F. Optimization o f Competence for New Escherichia coli Strains If the ultimate goal of a transformation experiment requires the use of a strain ofE. coli that has not been described here, nor in previous reports by Hanahan, 3'5 it may be necessary to determine which transformation protocol is most suited for the strain. It is first recommended that trial transformations be performed on the new strain using Protocols 5 (CCMB based) and 6 (TFB based). For the CCMB protocol cells should be grown without added magnesium (in SOB-Mg), whereas for the TFB-based version complete SOB medium is recommended. In addition, with the TFB version, the procedure can be tried without DMSO, without DTT, or without both. Both the density at which the cells are collected following their growth in SOB medium ( + / - Mg2÷) and the temperature at which the cells are grown can be varied. Regarding growth temperature, based on recent results one could consider testing new strains over a range from 18° to 37°. 5a'Sb If neither of these protocols gives adequate competence, the PEG/DMSO one-step procedure (Protocol 8) could be tried, as well as other variations on transformation buffers given by Hanahan. 3 Finally, electroshock transformation can be assessed,using the procedure given below (Protocol 10) or the variations suggested in Section V,C. It is our experience that most E. coli strains can be rendered competent to at least 10 7 transformants//xg, given a serious optimization process. However, unless the intended purpose specifically requires a specific new strain, the
14]
PLASMID TRANSFORMATION
83
strains recommended in Table II should be considered, as they have proved to be reliable hosts for plasmid tranformation.
IV. Induction o f Competence with Electroshock Treatment The treatment of cells with a brief pulse of high-voltage electricity has been found to permeabilize them toward entry of a variety of macromolecules. 1°'1~ It is presumed that the discharge of a voltage potential across a field which includes cells transiently depolarizes their membranes and induces pores which can be entry points for macromolecules. Such electroshock treatment has recently been shown to be applicable to competence induction of E. coli and other bacteria. For E. coli, electroshock transformation is the most efficient method available and approaches the theoretical maximum of 100% cell transformation frequencies. Efficiencies of greater than 101° transformants/ttg of pUC18/19 have been reported, 12,~3 and greater than 80% of all cells can be transformed by this method. More recent improvements in electronics, E. coli strains, and procedures have increased the transformation efficiency up to 5 × 10~°, as is described below) 4 For other bacteria, electroporation is the only method presently available for the efficient introduction of plasmid DNA. The exposure of a dense suspension of bacterial cells and plasmid DNA to a high-strength electric field of short duration has been found to induce DNA uptake and to elicit competence for stable plasmid transformation. The generation of an electroshock is usually accomplished by the discharge of a high-voltage capacitor through a mixture of bacterial cells and DNA suspended between two electrodes. The pulse length of the capacitor discharge can be varied by increasing the capacitor size or the resistance in the circuit itself, which includes the mixture of cell suspension and DNA. A parallel resistor can also be used to modulate the resistance of the electroshock circuit. The time of the electric current pulse (the shock) is described by a decay time constant ~', which corresponds to the time at which the voltage has dropped to approximately 37% of its original value. The time constant of the electroshock is determined by the product 0" = RC) of the resistance R (both of the cell/DNA mixture and any parallel resistor) and the capacitance C of the circuit through which the electric field is being discharged. 10 D. Knight, in "Techniques in Cellular Physiology" (P. F. Baker, ed.), pp. 1-20. Elsevier, Amsterdam, 1981. ii E. Neumann, M. Schaefer-Ridder, Y. Wang, and P. Hofschneider, EMBO J. 7, 841 (1982). 12 W. Dower, W. Miller, and C. Ragsdale, Nucleic Acids Res. 16, 6127 (1988). 13 W. Calvin and P. Hanawalt, J. Bacteriol. 170, 2796 (1988). 14 M. Smith, J. Jessee, T. Landers, and J. Jordan, Focus 12, 38 (1990).
84
[4]
Escherichia coli AND Salmonella typhimurium
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