Gene, 3 (1978) 279--292 279 © Elsevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
FACTORS AFFECTING THE TRANSFORMATION OF Escherichia coil
STRAIN X1776 BY pBR322 PLASMID DNA (Recombinant DNA; molecular cloning; cycloserine; vector DNA uptake) MICHAEL V. NORGARD, KIRSTEN KEEM and JOHN J. MONAHAN
Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 (U.S.A.) (Received February 16th, 1978) (Accepted April 14th, 1978) m
SUMMARY
The susceptibility of E. coli strain X1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5 . 0 - 6 . 0 • 107 cfu/ml, followed by washing twice in cold 0.1 M NaCI + 5 mM MgCI2 + 5 mM Tris, pH 7.6. Cells grown in the presence of V~ycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg 2÷ ions in washing and CaCI2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25--7.75 and a cell-to-DNA ratio of about 1.6. l 0 s cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCI2 in 250 mM KCI + 5 mM MgC12 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tc r transformants. In our hands, heat shocks at 37°C or 42°C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cel~ + DNA mixtures were plated on precooled (4°C) Tc agar plates. The efficiency of plating was optimum when only 5 , 1 of cell + DNA mixture was spread per plate, suggesting that non-viable background × 1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of × 1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli x 1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 107 transformants//~g pBR322 plasmid DNA. Abbreviations: cfu, colony-forming units; Cyc; D~ycloserine; DAP, diaminopimelic acid; Nx, nalidixic acid; To, tetracycline; Tcr, tetracycline-resistant.
280
INTRODUCTION
During our studies involving cloning total populations of mRNA sequences from early mouse embryos (using the plasmid pBR322), we initially observed a considerable amount of variability in the uptake of DNA by the NIH-certified EK2 host X1776. We had used the "CaCI2 method" for rendering Escherichia coli susceptible to transformation with plasmid DNA as described by Cohen et al. (1972). This method, which is essentially the same as that described by Mandel and Higa (1970) for rendering E. coil susceptible to transfection by purified phage DNA, has been widely used with E. coli, and has recently been extended for transformation of other bacterial hosts such as 8almonel!a typhimurium (Lederberg and Cohen, 1974) and Staphylococcus aureus (Lindberg et al., 1972). The method involves (i) several successive washes of the cells in cold NaCI and CaCI2 solutions, (ii) incubation in CaCI2 solution at 0°C with the DNA, and (iii) a short "heat shock" step at a higher temperature before plating the cells out in growth medium. The E. coli strain X1776 (Curtiss et al., 1976) was engineered to be particularly sensitive to ordinary media and conditions other than those selected specifically for its survival. Since we and others have experienced difficulties obtaining transformations with this strain, it seemed reasonable to reappraise the CaC12 treatment conditions developed for use with hardier E. coli strains. This report describes a number of parameters that have been optimized for the transformation of x1776 using the plasmid pBR322. MATERIALS AND METHODS
(a) Bacterial strains E. coli strain RR1 F - p r o leu thi/acY Strr r- k m- k (Bolivar et al., 1977) harboring the plasmid pBR322 (Bolivar et al., 1977) was used in the propagation of pBR322 plasmid donor DNA. E. coli K12 strain X1776 FtonA53 dapD8 minA1 supE42 A40(ga/-uvrB) k- minB2 nalA25 thyA57 metC65 A29(bioH-asd) cycB2 cycA1 HsdR2 was provided by R. Curtiss III and used as the recipient strain in transformation. E. coli strain C600 (Appleyard, 1954) was used as a source of E. coli bulk DNA. (b) Media and growth conditions E. coli RR1 was cultivated in M9 glucose minimal broth medium (Adams, 1959) and maintained on trypticase soy agar. The composition of media for optimal growth and transformation of strain x 1776 was found to be critical and was prepared as follows: Each liter of "L broth" contained 5 g Bactoyeast extract (Difco), 10 g Bacto-tryptone (Difco), 1 0 g NaCI, and was further supplemented with 1/~g/ml d-biotin (Sigma), 100/~g/ml DL-diamino. pimelic acid (DAP) (Calbiochem No. 2815), 2 0 / ~ g / ~ thymidine (Sigma T-9250), and 0.08% (w/v) glucose. After autoclaving under standard conditions for 20 rain, the medium was allowed to cool to 50 °C before the addi-
281
tion of filter-sterilized (Millipore, 0.45/~m) Cyc (Sigma) to a final concentration of 20 ~g/ml. This broth was routinely used for the large-scale cultivation of X1776 cells to be used in transformation experiments. However, L broth containing 50/~g/ml Nx (P-L Biochemicals, Inc.) rather than Cyc was used in the maintenance and preparation of overnight inocula of × 1776 cells. L agar plates were prepared by adding 1.5% Bacto-agar to L broth before autochving. For the selection of tetracycline-resistav.t (Tc r) transformants, L agar was allowed to cool to 50°C after autoclaving and was supplemented with filtersterilized Tc to a final concentration of 12.5/~g/ml and Cyc to a final concentration of 20 ~g/ml. Unless otherwise specified, bacterial broth cultures were incubated at 37°C while shaking at 125 rev./min. The pH of the growth media did not change more than 0.1 pH unit during the growth conditions. Bacterial stock cultures were maintained on agar medium stored at 4°C, and transferred monthly to ensure viability.
(c) Isolation o f pBR322 plasmid donor DNA and E. coU bulk DNA Amplification of pBR322 plasmid in E. coil RRI was carried out as described by Clewell et al. (1972). Briefly, RR1 (harboring pBR322) was grown in M9 glucose medium. When the absorbance at 550 nm (Ass0) of the culture reached 0.3, 100/~g/ml chloramphenicol (Sigma No. C0378) was added followed by additional incubation for 12 h. Isolation and purification of the plasmid DNA was as described by Clewell and Helinski (1970). The DNA was further purified by CsCI density gradient centrifugation (Radloff et al., 1967). Bulk E. coli DNA was isolated from strain C600 using the c h l o r o f o ~ isoamyl alcohol method (Marmur, 1961).
(d) Transformation The optimized conditions for transformation were as follows: An overnight culture of strain X1776 (in L broth + Nx) was diluted 1:100 in fresh L broth (+ Cyc) prewarmed to 37°C. The culture was then incubated under standard conditions until the Ass0 of the culture reached 0.28--0.32 (approx. 5.0--6.0 • 10 ~ cfu/ml). At this time, cells from each 50 ml of culture were harvested by centrifugation in the cold for 10 min at 2500 X g and washed twice by centrifugation for 5 rain in 25 ml cold 0.1 M NaCI + 5 mM MgCI2 + 5 mM Tris--HCl, pH 7.6. After treatment for 25 min on ice in 20 ml of 100 mM CaCI2 + 250 mM KCI + 5 mM MgCI2 + 5 mM Tris--HCl, pH 7.6, cells were centrifuged for 5 rain in the cold and resuspended in a total volume of 0.4 ml of the CaCI2 buffer. 0.2 ml of cell suspension was then mixed with 0.1 ml pBR322 plasmid DNA stock solution (30 ng/ml in 0.1 M NaCI + 5 mM Tris--HCl, pH 7.6) in 1.5 ml polypropylene microtest tubes (Bio-Rad Laboratories Inc.) and incubated on ice for a total of 60 rain, with gentle mixing every 10 rain. 5--25/~1 was then plated directly on precooled (4°C) Tc agar selective medium, which was prepared the same day or one day prior to use, followed by incubation of the plates at 37°C for 48 h. Since an excess of
282
cells was employed in cell + DNA mixtures, transformants were scored as the number of Tc r transformants per ng of pBR322 plasmid DNA. Also, because not all experiments were performed using optimal conditions for transformation, transformation frequencies were expressed as Relative Transformation (%) = percent of maximum transformation obtained within each experiment. Viable cell count assays on eel] + DNA mixtures (after 60 min on ice) were performed by se~ally diluting 10-fold in cold L broth and plating 0.1 ml on L agnr followed by incubation at 37°C for 48 h. RESULTS
Several individual variables were separately investigated. The findings of these experiments are grouped below in terms of major parameters studied. Effect of culture conditions. To determine the optimal cell density for transformation, × 1776 cells in L broth culture were harvested at various intervals. It was found that cells harvested at 5 . 0 - 6 . 0 - 107 cfu/ml (Ass0 0.280.32) produced the maximum number of transformants (Fig. 1). It is apparent from the curve in Fig. I that cells grown to densities outside this narrow range yielded a significantly lower frequency of transformation. It was also observed that the initial inoculum size for the starting culture influenced the trans. formabflity of X1776 cells. For example, the overnight inoculum which was routinely diluted 1/100 in fresh, prewarmed (37°C) L broth medium, gave best results. Inocula diluted 1/20 yielded X1776 cells which transformed only about 50% as well, even when the cells were harvested at the optimum culture density. We concluded that cultures should be inoculated as low as practically possible. I
'
I "I'I'
I00 eo
t l 2
,
I , i,l,l~ 4 6810
CFU/ml xlO"7
Fig. 1. Effect of cell density on the transformation of x1776. Cells were harvested at various growth stages and tested for their susceptibility to transformation by pBR322 plasmld DNA. Each aliquot of cell suspension was n o r ~ i z e d to 1.7-107 cf-u/ml. This was dm~e as follows: An Ass0 vs. cfu/ml s t a n ~ d curve was made. From the As~0 of the growth culture, a calculated quantity of cells was removed and mixed with fresh medium to obtain 1.7-10 ~ cfu/ml.
283
Transformation levels were found to be markedly better when X1776 cells were grown in and plated on media which contained 20/~g/ml Cyc rather than 50/~g/ml Nx. The difference was about a factor of 4. Consequently, Cyc was added to all media used in transformation experiments with X1776. Effect of cell washings. Table I summarizes the effect of cell washings on the transformation of X1776. Cells harvested from 50 ml of culture and washed twice in 25 ml cold NaCI buffer showed optimal transformation, and cells washed three times exhibited only slightly lower transformation levels. Cells washed once or not at all yielded transformation levels some 40.3% and 84.4% lower, respectively, than those twice-washed. The transformation frequencies apparently correlated to some degree with cell viabilities after CaCl2 treatment and after incubation on ice for 60 min in the presence of transforming plasmid DNA. Unwashed cells showed only a 64.3% viability as compared with twice-washed cells, and cells washed once demonstrated a viability of 78.6% when compared with twice-washed cells. Thrice-washed cells showed a viability of 92.9%, which may have been partially due to cell loss during centrifugation washing procedures. Effect o f Ca2+ and Mg 2+ ions. The abilities of various Ca 2+ ion concentrations to promote the transformation of × 1776 were examined. In each case washed cells from 50 ml of culture were suspended in 20 ml of various CaC12 concentrations ranging from 25--150 mM CaC12 (in KC1 + MgCl2 + Tris buffer), incubated on ice for 25 min, centrifuged, and suspended in 0.4 ml of the same buffer. Then 0.2 ml of the cell mixture was incubated for 60 min on TABLE I EFFECT OF CELL WASHING ON TRANSFORMATION OF X1776 Cells from each 50 ml of culture were harvested and washed for an appropriate number of times by centrifugation in 25 ml cold 0.1 MNaCI + 5 mM MgCI~ + 5 mM Tris--HCl, pH 7.6. Cells were then prepared for transformation as described in MATERIALS AND METHODS. Viabilities of x1776 cells in cell + DNA reaction mixtures after a 60 min incubation on ice were determined by serially diluting 10-fold in cold L broth and plating 0.1 ml on L agar followed by incubation at 37°C for 48 h. Relative transformation (%) levels, which were monitored in each case by the appearance of Tc r clones on Tc agar, were calculated as the percent of maximum transformation obtained within the experiment (i.e., from twice-washed cells). Number of cell washes
Viability (cfu/ml) of cell + DNA mixture
% of maximum viability
Relative transformation
64.3 78.6 100.0 92.9
15.6 59.7 100.0 93.1
after 60 min on ice (.10 °) 0 1 2 3
0.9 1.1 1.4 1.3
(%)
284
ice with 0.1 ml of the DNA solution. As shown in Fig. 2, maximum transformation was achieved when cells were treated with 100 mM CaCI2. However, this range was quite broad with 94% transformation at 70 and 150mM CcC12. ~ effect of M ~ ÷ ions was ~i~o investigated. Fig. 3 shows that a d d i ~ 5 m M ' - b ~ 2 to both--°NaCl washes and ~he CaCI2 bufferinereased the frequency of X1776 transformation more than 3-fold. MgCI2 levels below about 4raM or above about 7.5 mM appeared to stimulate transformation to alesser degree.
~)
so
i--
le~, 2
I
,
4
!
,
6
!
,
8
I-
I I0
15
mM CaCI2 x 10"1
1;';,, 9. = , e - . ~ of CaCI concentration in promoting the transfonne~,ion of x1776. Approx.
4".'~'o10'c~'f=were tre;ted with a range of CaCI, concentrations as .*
bed in the text,
followed by treatment with pBR322 DNA to determine an optimum concentration of CaCI2 to facilitate transformation. 100% relative transformat!.on = 1.9. • 106 cloncs/~g pBR322 DNA (plating 50 ~1 of the cell + DNA mixture per dish. No Mg 2÷ was present during this experiment).
~) 80 60 im
:
FACTORS AFFECTING THE TRANSFORMATION OF Escherichia coil
STRAIN X1776 BY pBR322 PLASMID DNA (Recombinant DNA; molecular cloning; cycloserine; vector DNA uptake) MICHAEL V. NORGARD, KIRSTEN KEEM and JOHN J. MONAHAN
Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110 (U.S.A.) (Received February 16th, 1978) (Accepted April 14th, 1978) m
SUMMARY
The susceptibility of E. coli strain X1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5 . 0 - 6 . 0 • 107 cfu/ml, followed by washing twice in cold 0.1 M NaCI + 5 mM MgCI2 + 5 mM Tris, pH 7.6. Cells grown in the presence of V~ycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg 2÷ ions in washing and CaCI2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25--7.75 and a cell-to-DNA ratio of about 1.6. l 0 s cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCI2 in 250 mM KCI + 5 mM MgC12 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tc r transformants. In our hands, heat shocks at 37°C or 42°C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cel~ + DNA mixtures were plated on precooled (4°C) Tc agar plates. The efficiency of plating was optimum when only 5 , 1 of cell + DNA mixture was spread per plate, suggesting that non-viable background × 1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of × 1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli x 1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 107 transformants//~g pBR322 plasmid DNA. Abbreviations: cfu, colony-forming units; Cyc; D~ycloserine; DAP, diaminopimelic acid; Nx, nalidixic acid; To, tetracycline; Tcr, tetracycline-resistant.
280
INTRODUCTION
During our studies involving cloning total populations of mRNA sequences from early mouse embryos (using the plasmid pBR322), we initially observed a considerable amount of variability in the uptake of DNA by the NIH-certified EK2 host X1776. We had used the "CaCI2 method" for rendering Escherichia coli susceptible to transformation with plasmid DNA as described by Cohen et al. (1972). This method, which is essentially the same as that described by Mandel and Higa (1970) for rendering E. coil susceptible to transfection by purified phage DNA, has been widely used with E. coli, and has recently been extended for transformation of other bacterial hosts such as 8almonel!a typhimurium (Lederberg and Cohen, 1974) and Staphylococcus aureus (Lindberg et al., 1972). The method involves (i) several successive washes of the cells in cold NaCI and CaCI2 solutions, (ii) incubation in CaCI2 solution at 0°C with the DNA, and (iii) a short "heat shock" step at a higher temperature before plating the cells out in growth medium. The E. coli strain X1776 (Curtiss et al., 1976) was engineered to be particularly sensitive to ordinary media and conditions other than those selected specifically for its survival. Since we and others have experienced difficulties obtaining transformations with this strain, it seemed reasonable to reappraise the CaC12 treatment conditions developed for use with hardier E. coli strains. This report describes a number of parameters that have been optimized for the transformation of x1776 using the plasmid pBR322. MATERIALS AND METHODS
(a) Bacterial strains E. coli strain RR1 F - p r o leu thi/acY Strr r- k m- k (Bolivar et al., 1977) harboring the plasmid pBR322 (Bolivar et al., 1977) was used in the propagation of pBR322 plasmid donor DNA. E. coli K12 strain X1776 FtonA53 dapD8 minA1 supE42 A40(ga/-uvrB) k- minB2 nalA25 thyA57 metC65 A29(bioH-asd) cycB2 cycA1 HsdR2 was provided by R. Curtiss III and used as the recipient strain in transformation. E. coli strain C600 (Appleyard, 1954) was used as a source of E. coli bulk DNA. (b) Media and growth conditions E. coli RR1 was cultivated in M9 glucose minimal broth medium (Adams, 1959) and maintained on trypticase soy agar. The composition of media for optimal growth and transformation of strain x 1776 was found to be critical and was prepared as follows: Each liter of "L broth" contained 5 g Bactoyeast extract (Difco), 10 g Bacto-tryptone (Difco), 1 0 g NaCI, and was further supplemented with 1/~g/ml d-biotin (Sigma), 100/~g/ml DL-diamino. pimelic acid (DAP) (Calbiochem No. 2815), 2 0 / ~ g / ~ thymidine (Sigma T-9250), and 0.08% (w/v) glucose. After autoclaving under standard conditions for 20 rain, the medium was allowed to cool to 50 °C before the addi-
281
tion of filter-sterilized (Millipore, 0.45/~m) Cyc (Sigma) to a final concentration of 20 ~g/ml. This broth was routinely used for the large-scale cultivation of X1776 cells to be used in transformation experiments. However, L broth containing 50/~g/ml Nx (P-L Biochemicals, Inc.) rather than Cyc was used in the maintenance and preparation of overnight inocula of × 1776 cells. L agar plates were prepared by adding 1.5% Bacto-agar to L broth before autochving. For the selection of tetracycline-resistav.t (Tc r) transformants, L agar was allowed to cool to 50°C after autoclaving and was supplemented with filtersterilized Tc to a final concentration of 12.5/~g/ml and Cyc to a final concentration of 20 ~g/ml. Unless otherwise specified, bacterial broth cultures were incubated at 37°C while shaking at 125 rev./min. The pH of the growth media did not change more than 0.1 pH unit during the growth conditions. Bacterial stock cultures were maintained on agar medium stored at 4°C, and transferred monthly to ensure viability.
(c) Isolation o f pBR322 plasmid donor DNA and E. coU bulk DNA Amplification of pBR322 plasmid in E. coil RRI was carried out as described by Clewell et al. (1972). Briefly, RR1 (harboring pBR322) was grown in M9 glucose medium. When the absorbance at 550 nm (Ass0) of the culture reached 0.3, 100/~g/ml chloramphenicol (Sigma No. C0378) was added followed by additional incubation for 12 h. Isolation and purification of the plasmid DNA was as described by Clewell and Helinski (1970). The DNA was further purified by CsCI density gradient centrifugation (Radloff et al., 1967). Bulk E. coli DNA was isolated from strain C600 using the c h l o r o f o ~ isoamyl alcohol method (Marmur, 1961).
(d) Transformation The optimized conditions for transformation were as follows: An overnight culture of strain X1776 (in L broth + Nx) was diluted 1:100 in fresh L broth (+ Cyc) prewarmed to 37°C. The culture was then incubated under standard conditions until the Ass0 of the culture reached 0.28--0.32 (approx. 5.0--6.0 • 10 ~ cfu/ml). At this time, cells from each 50 ml of culture were harvested by centrifugation in the cold for 10 min at 2500 X g and washed twice by centrifugation for 5 rain in 25 ml cold 0.1 M NaCI + 5 mM MgCI2 + 5 mM Tris--HCl, pH 7.6. After treatment for 25 min on ice in 20 ml of 100 mM CaCI2 + 250 mM KCI + 5 mM MgCI2 + 5 mM Tris--HCl, pH 7.6, cells were centrifuged for 5 rain in the cold and resuspended in a total volume of 0.4 ml of the CaCI2 buffer. 0.2 ml of cell suspension was then mixed with 0.1 ml pBR322 plasmid DNA stock solution (30 ng/ml in 0.1 M NaCI + 5 mM Tris--HCl, pH 7.6) in 1.5 ml polypropylene microtest tubes (Bio-Rad Laboratories Inc.) and incubated on ice for a total of 60 rain, with gentle mixing every 10 rain. 5--25/~1 was then plated directly on precooled (4°C) Tc agar selective medium, which was prepared the same day or one day prior to use, followed by incubation of the plates at 37°C for 48 h. Since an excess of
282
cells was employed in cell + DNA mixtures, transformants were scored as the number of Tc r transformants per ng of pBR322 plasmid DNA. Also, because not all experiments were performed using optimal conditions for transformation, transformation frequencies were expressed as Relative Transformation (%) = percent of maximum transformation obtained within each experiment. Viable cell count assays on eel] + DNA mixtures (after 60 min on ice) were performed by se~ally diluting 10-fold in cold L broth and plating 0.1 ml on L agnr followed by incubation at 37°C for 48 h. RESULTS
Several individual variables were separately investigated. The findings of these experiments are grouped below in terms of major parameters studied. Effect of culture conditions. To determine the optimal cell density for transformation, × 1776 cells in L broth culture were harvested at various intervals. It was found that cells harvested at 5 . 0 - 6 . 0 - 107 cfu/ml (Ass0 0.280.32) produced the maximum number of transformants (Fig. 1). It is apparent from the curve in Fig. I that cells grown to densities outside this narrow range yielded a significantly lower frequency of transformation. It was also observed that the initial inoculum size for the starting culture influenced the trans. formabflity of X1776 cells. For example, the overnight inoculum which was routinely diluted 1/100 in fresh, prewarmed (37°C) L broth medium, gave best results. Inocula diluted 1/20 yielded X1776 cells which transformed only about 50% as well, even when the cells were harvested at the optimum culture density. We concluded that cultures should be inoculated as low as practically possible. I
'
I "I'I'
I00 eo
t l 2
,
I , i,l,l~ 4 6810
CFU/ml xlO"7
Fig. 1. Effect of cell density on the transformation of x1776. Cells were harvested at various growth stages and tested for their susceptibility to transformation by pBR322 plasmld DNA. Each aliquot of cell suspension was n o r ~ i z e d to 1.7-107 cf-u/ml. This was dm~e as follows: An Ass0 vs. cfu/ml s t a n ~ d curve was made. From the As~0 of the growth culture, a calculated quantity of cells was removed and mixed with fresh medium to obtain 1.7-10 ~ cfu/ml.
283
Transformation levels were found to be markedly better when X1776 cells were grown in and plated on media which contained 20/~g/ml Cyc rather than 50/~g/ml Nx. The difference was about a factor of 4. Consequently, Cyc was added to all media used in transformation experiments with X1776. Effect of cell washings. Table I summarizes the effect of cell washings on the transformation of X1776. Cells harvested from 50 ml of culture and washed twice in 25 ml cold NaCI buffer showed optimal transformation, and cells washed three times exhibited only slightly lower transformation levels. Cells washed once or not at all yielded transformation levels some 40.3% and 84.4% lower, respectively, than those twice-washed. The transformation frequencies apparently correlated to some degree with cell viabilities after CaCl2 treatment and after incubation on ice for 60 min in the presence of transforming plasmid DNA. Unwashed cells showed only a 64.3% viability as compared with twice-washed cells, and cells washed once demonstrated a viability of 78.6% when compared with twice-washed cells. Thrice-washed cells showed a viability of 92.9%, which may have been partially due to cell loss during centrifugation washing procedures. Effect o f Ca2+ and Mg 2+ ions. The abilities of various Ca 2+ ion concentrations to promote the transformation of × 1776 were examined. In each case washed cells from 50 ml of culture were suspended in 20 ml of various CaC12 concentrations ranging from 25--150 mM CaC12 (in KC1 + MgCl2 + Tris buffer), incubated on ice for 25 min, centrifuged, and suspended in 0.4 ml of the same buffer. Then 0.2 ml of the cell mixture was incubated for 60 min on TABLE I EFFECT OF CELL WASHING ON TRANSFORMATION OF X1776 Cells from each 50 ml of culture were harvested and washed for an appropriate number of times by centrifugation in 25 ml cold 0.1 MNaCI + 5 mM MgCI~ + 5 mM Tris--HCl, pH 7.6. Cells were then prepared for transformation as described in MATERIALS AND METHODS. Viabilities of x1776 cells in cell + DNA reaction mixtures after a 60 min incubation on ice were determined by serially diluting 10-fold in cold L broth and plating 0.1 ml on L agar followed by incubation at 37°C for 48 h. Relative transformation (%) levels, which were monitored in each case by the appearance of Tc r clones on Tc agar, were calculated as the percent of maximum transformation obtained within the experiment (i.e., from twice-washed cells). Number of cell washes
Viability (cfu/ml) of cell + DNA mixture
% of maximum viability
Relative transformation
64.3 78.6 100.0 92.9
15.6 59.7 100.0 93.1
after 60 min on ice (.10 °) 0 1 2 3
0.9 1.1 1.4 1.3
(%)
284
ice with 0.1 ml of the DNA solution. As shown in Fig. 2, maximum transformation was achieved when cells were treated with 100 mM CaCI2. However, this range was quite broad with 94% transformation at 70 and 150mM CcC12. ~ effect of M ~ ÷ ions was ~i~o investigated. Fig. 3 shows that a d d i ~ 5 m M ' - b ~ 2 to both--°NaCl washes and ~he CaCI2 bufferinereased the frequency of X1776 transformation more than 3-fold. MgCI2 levels below about 4raM or above about 7.5 mM appeared to stimulate transformation to alesser degree.
~)
so
i--
le~, 2
I
,
4
!
,
6
!
,
8
I-
I I0
15
mM CaCI2 x 10"1
1;';,, 9. = , e - . ~ of CaCI concentration in promoting the transfonne~,ion of x1776. Approx.
4".'~'o10'c~'f=were tre;ted with a range of CaCI, concentrations as .*
bed in the text,
followed by treatment with pBR322 DNA to determine an optimum concentration of CaCI2 to facilitate transformation. 100% relative transformat!.on = 1.9. • 106 cloncs/~g pBR322 DNA (plating 50 ~1 of the cell + DNA mixture per dish. No Mg 2÷ was present during this experiment).
~) 80 60 im
:
