Gene, 96 (1990) 23-28 Elsevier

GENE 03769

23

High efficiency transformation of Escher ichia col i with plasmids

(Competent cell; strain DH5; pBR322 vector; simple and efficient method (SEM); cDNA library; electroporation; frozen- stock; stability)

Hiroaki Inoue', Hiroshi Nojima b and Hiroto 0kayama b

a Research Department, Turuga Enzyme Plant, Toyob~ Co, Ltd., Turuga City, Fukui-ken 914 (Japan) Tel. (0770)22 7643, and b Department of Molecular Genetics, Research Institute for Microbial .Oisease~, Osaka University, Yamadaoka 3-!, Suita City, Osaka 565 (Japan)

Received by H. Yoshikawa: 26 April 1990 Revised: 4 June 1990 Accepted: 15 June 1990

SUMMARY

We have re-evaluated the conditions for prepmmg competent Escherichia coil cells and established a simple and efficient method (SEM)for plasmid transfection. Cells (DH5, JMI09 and HB101) prepared by SEM are extremely competent for transformation (1-3 x 109 efu//~g of pBR322 DNA), and can be stored in liquid nitrogen for at least 40 days without loss of competence. Unlike electroporation, transformation using these competent cells is affected minimally by salts in DNA preparation. These competent cells are particularly useful for construction of high-complexity cDNA libraries with a minimum expenditure of mRNA.

INTRODUCTION

Recent advances in recombinant DNA techniques have made it possible to isolate a eDNA copy of a gene by complementation of a particular defect in cells without information of its gene product. The efficacy ofthis strategy, which is called 'expression cloning' or 'complementation

Correspondence to: Dr. H. Okayama, Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3-1, Suita City, Osaka 565 (Japan) Te!.(06)8753980; Fax (06) 875 5192.

Abbreviations: Ap, ampicillin; BES, N,N-bis(2-hydroxyethyl)-2-amino- ethanesulfonic acid; bp; base pair(s); cfu, colony-forming unit(s); DMSO, dimethyl sulfoxide; FTB, freeze-thaw buffer (see section c and Fig. 1 legend); Hepes, N-(2-hydroxymethyl)piperazine-N'-2-ethanesul- fonic acid; kb, kilobase(s) or 1000 bp; LB, Luria-Bertani (medium); MOPS, 3-(N-morpholino)propanesulfonic acid; nt, nucleotide(s); Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); SEM, simple and efficient method for transformation; SOB; 2% Bacto tryptone/0.5% yeast extract/10 mM NaCI/2.5 mM KCI/10 mM MgCI2/10 mM MgSO4; SOC, SOB with 20 mM glucose; TB, transformation buffer (see section a); TE, 10 mM Tris. HCI (pH7.5)/1 mM EDTA.

cloning', largely depends on the quality of the eDNA library used for the experiments. We have hitherto developed a vector system that permits construction of full-length eDNA libraries and subsequent screening based on pheno- typic expression of the eDNA inserts in mammalian cells (Okayama and Berg, 1982; 1983; Okayama et al., 1987; Chen and Okayama, 1987). To screen for very rare eDNA clones, a high complexity eDNA library consisting of at least 5-10 x 106 independent clones often needs to be ~tm- structed with a limited amount of mRNA. This task could not be achieved if highly competent E. coli cells were not available. We have used the protocol described by Hanahan (1983) with a slight modification (Okayama et al., 1987). This Hanahan protocol is one of the best chemical methods presently available) whichwas developed to attain the maxi- mum transformation effieiencies after a great deal of studies (Taketo, 1972; Taketo and Kuno, 1974; Lederberg, 1974; Kretschmer et al., 1975; Enea et al., 1975: Dagert and Ehrlich, 1979; Morrison, 1979; Jones etal., 1981; Bergmans et al., 1981) since the first demonstration of Ca 2 + -dependent DNA transfer into E. coli by Mandel and Higa (1970). However, preparation of cells with a con-

0378-1119/90/$03.50 1990 Elsevier Science Publishers B.V. (Biomedical Division)

24

sistently high transformation efficiency is difficult in general. Moreover, frozen competent cells often rapidly lose their competence during storage, particularly when the final liga- tion product in the process of eDNA-library construction is used for transfection. To circumvent these problems, we systematically optimized each condition and parameter for the prepacation of E. coli competent cells.

The aim of the present study was to develop SEM for the preparation of such cells with an extremely high transform- ing frequency (1-3 x 10 9 cfu//2g of plasmid DNA).

RESULT AND DISCUSSION

(a) Standard protocol for the preparation of competent cells by SEM

Media (SOB, SOC and LB) were prepared as described by Hanahan (1983). For selection of transformed E. coli, LB plates containing 50/~g Ap/ml were used. To make TB (10 mM Pipes/55 mM MnCI2/15 mM CaC12/250mM KCI), all the components except for MnCI 2 were mixed and the pH was adjusted to 6.7 with KOH. Then, MnCI 2 was dissolved, the solution was sterilized by filtration through a preripsed 0.45 pm filter unit and stored at 4C. All salts were added as solids.

Frozen stock (in LB/7~o DMSO) DH5 cells were thawed, streaked on an LB agar plate, and cultured over- night at 37C. About ten to twelve large (2-3 mm in diameter) colonies were isolated with a plastic loop, inocu- lated to 250 ml of SOB medium in a 2-liter flask, and grown to an A6o o of 0.6 at 18C, with vigorous shaking (200-250 rpm). The flask was removed from the incubator and placed on ice for 10 min. The culture was transferred to a 500ml centrifuge bottle and spun at 2500 x g 3~00 rpm in Beckmann J-6B centrifuge) for 10 min at 4C. The pellet was resuspended in 80ml of ice-cold TB, incubated in an ice bath for 10 min, and spun down as above. The cell pellet was gently resuspended in 20 ml of TB, and DMSO was added with gentle swirling to a final concentration of 7~o. After incubating in an ice bath for 10 min, the cell suspension was dispensed by 1-2 ml into tissue-culture cell-freezing tubes and immediately chilled by immersion in liquid nitrogen. The frozen competent cells were stored in liquid nitrogen for at least one month without a detectable loss of competence.

(b) Standard protocol for SEM transformation A tube of competent cells was thawed at room tempera-

ture, dispensed by 200/~1 into 15 ml polypropylene tubes (Greiner, F.R.G.) and placed in an ice bath. Glass tubes should not be used since they largely decrease (tenfold less) tlae competence. Usually, 1-5/~1 of the plasmid zeMtion was added to each tube, and the cells were incubated in an

ice bath for 30 min. They were then heat-pulsed without agitation at 42C for 30 s and transferred to an ice bath. After 0.8 ml of SOC was added, the tubes were placed in a 37 C incubator and shaken vigorously for 1 h. A desired portion of the mixture was tranferred into a 5 ml poly- propylene or glass tube, mixed with 3 ml of melted LB soft agar preincubated at 47C, and poured on LB plates containing 50 #g Ap/ml. Colonies were counted after over- night incubation at 37 o C.

(c) Optimization of transformation buffer To be well suited for construction of high-complexity

libraries, competent cells must meet two requirements: (i)high-frequency transformation with ligated DNA; (ii) ability to be stored frozen in liquid nitrogen without loss of competence. To find the best conditions for preparing competent cells, we began by optimizing FTB (Hanahan, 1983). To simplify our strategy, we first restricted the plas- mid DNA and the E. coli strain to pBR322 and DH5, respectively. The E. coli DH5 strain (supE44 hsdRl7 (r~ ,m~ ) recAI endA 1 gyrA96 thi-I relA I) appears to be suitable as a host for generating cDNA libraries, since plasmids containing homologous sequences are stable in this strain owing to its recA 1 genotype. Moreover, this strain is efficiently transformed with large plasmids because it is defective in endonuclease I. These properties are important when full-length cDNA libraries containing more than 6-7 kb inserts are to be constructed.

First, we examined the concentration of the components and pH values of FTB without significant improvements. Second, we examined the amount ofglycerol added to FTB and found that, in our hands, glycerol (a glass-distilled grade from BRL) in FTB was highly inhibitory to com- petence of DH5 (Fig. la). In the course of this investigation, we found that all salts, e~xcept KCI, CaCI2, MnCI2 and K.acetate, could be removed from FTB. Salts such as hexamine-Co chloride, COSO4, LiCI and RbCl were found to be inhibitory rather than stimulatory to transformation. Therefore, they were removed from the transformation buffer. The pH value of the transformation buffer which has little influence on transformation efficiency in a range of 5.6-7.0, could not be raised more than 7.0 because of sedimentation of manganic salts at this a~d higher pH values. Hence, we chose pH 6.7.

Fig. lb shows that among buffers tested, Pipes yielded the highest competence. Therefore, K. acetate was replaced by Pipes or Hepes. After optimizing the pH (Fig. 2a) and salt concentrations (Fig. 2b-d) of this solution, we decided to use TB (see section a) as our standard transformation buffer. The optimum concentration of these omponents remained the same when 10 mM Pipes (pH 6.7) was replaced by 10 mM Hepes (pH 6.7). Competent cells pre- pareG ". y the standard transfection protocol (see section a;

25

1010

u P- lO 9

u . v-

g~

u 0 7 F-

106

0 10 20 1 2 3 4 s

Glycero l (%)

Fig. I. Effect of glycerol and buffers on transformation frequency (cfu/ /Jg of pBR322 DNA). (a)Effect of glycerol included in the FTB (Hana- han, 1983; 10 mM K.acetate/45 mM MnCl2/10 mM CAC12/3 mM [Co(NH~)2]CI3 pH6.4). Competent cells were stored for I h in liquid nitrogen. (h) Difference in transformation frequency when 10raM K. acetate (bar 1) in the modified FTB (see section e) was replaced by 10 mM Hepes (bar 2), 10 mM BES (bar 3), 10 mM MOPS (bar 4) or 10 mM Pipes (bar 5). In both experiments, cells (DH5) were grown at 18"C and harvested at an A6o o of 0.37. Each value for competence was an average of duplicate measurements.

200/tl of competent cells transfected with 10 pg pBR322 DNA), yielded a transformation frequency of 1-3 x 109 cfu//zg pBR322. This value corresponds to about one transformed cell per 70-200 plasmid DNA molecules.

(d) Opt imizat ion of cell growth

After testing various concentrations of the components of the medium for E. coil growth, we found that SOB and

SOC media (Hanahan, 1983) were suitable for obtaining optimal efficiency for our method. The optimum tempera- ture used for cell growth, however, was very different from Hanahan's (1983). As shown in Fig. 3, incubation at 18C, although it might take two days to reach a proper cell concentration, gave high competence in a wide range of cell concentrations at harvest. The optimum cell density was an A6o o of 0.75. On the other hand, incubation at 37C as described by Hanahan (1983) resulted in both a decrease in transformation frequency and a sharp dependence of com- petence on cell density. The optimum cell density at this temperature was an A6o o of 0.45 (Hanahan, 1983). Incuba- tion at room temperature (about 25 C) also gave results similar to incubation at 18 C, but with a slight decrease in competence (data not shown). Incubation at a temperature lower than 15C is impractical since growth is too slow. Use ofFTB (Hanahan, 1983), however, resulted in only low competences (2 x 107-2 x 10 s cfu/#g of pBR322) at any concentration of cells even after incubation at 18C.

(e) Optimizat ion of transfection steps

In our standard transformation protocol, frozen com- petent cells were thawed at room temperature and trans- ferred to an ice bath as soon as thawing was completed. Competent cells (0.2 ml) have been dispensed into poly- propylene of polystyrene tubes (Greiner, F.R.G.). The pBR322 DNA (10 pg each in 1 #1 of TE) was then added to the competent cells and the mixture incubated in an ice bath for 30 min. The volume of DNA solution (up to 20 #1) had little influence on transformation frequency (data not shown). The number of transformants increased in pro- portion to the amount of DNA up to 10 ng. Afterwards,

I: S~IO 9

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pH PIPES(mM) MnCI2(mM) KCI (raM) CaCl=(mM) A soo

Fig. 2. Influences ofpH (a) and concentrations of TB components (b-e) on transformation frequency (cfu/#g pBR322 DNA). One of the parameters in TB (see section a) was varied. The cells DH5 were grown at 18 C and harvested at an A6oo of 0A2. Transformation frequency at each point was obtained by duplicate measurements. Fig. 3. Effect of cell concentration at harvest and growth temperature on transformation frequency (cfu/#g of pBR322 DNA). Cells were grown at 37 C (D) or 18C (m), treated with standard TB and transfected following the standard protocol (see section a). Each point denotes an average value of duplicate measurements.

26

however, the ratio of transformants to the amount of used DNA decreased rapidly (data not shown).

To optimize heat-shock conditions, transfected cell sus- pensions were subjected to a heat pulse at 40 , 42 and 44C for 0-90 s. As shown in Fig. 4a, the highest number of transformants was yielded at 30 s. The temperature for the heat pulse was not critical, and even after heat shock at an extremely high temperature (52C) for 30 s, a similar transformation efficiency was obtained (Fig. 4b).

(f) Freezing competent cells and their stability The benefit of freezing competent cells is twofold.

Freezing increases the competence, and storage of frozen competent cells permits knowing their competence prior to use for precious transformation such as library construe- tion. DMSO is most widely used as a stabilizer of frozen cells and worked well for our competent cells. The optimum concentration of DMSO was 7~o for the cold shock and storage of the competent cells (Fig. 5a). Freezing in liquid nitrogen (cold shock) enhanced the transformation effi- ciency four- to fivefold. Frozen competent cells in the pres- ence of 7% DMSO were stable. As shown in Fig. 5b, cells retained their original conipetence even after a 40 days' storage in liquid nitrogen. Oxidation products of DMSO did not seem to be critical to our competent cells. We used DMSO from a bottle that had been opened many times and stored at room temperatu,e without a detectable decrease in effectiveness. DMSO from commercial sources other than MCB (optical grade) gave similar good results.

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Time (second) Temperature (c) Fig. 4. Effects of time (a) and temperature (b) of heat shock on transfor- mation frequency. Cells (DHS) were grown at 18 C, harvested at an A60o of 0.6I and treated with standard transformation buffer. Effect of time was assessed at three different temperatures; 40C (1), 42C (1"1) and 44C (O). Average values of duplicate measurements were plotted.

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' 3 ~ l e

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b

~ m . m ~ l ~ ' i ~ g '

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Fig. 5. Effects ofDMSO and stability ofcompetence. (a) Transfor~la~ion efficiency as a function of DMSO concentration. Transformation fre- quency was measured after thawing competent cells prepared by the standard transformation protocol except that the DMSO concentration was varied (see section b). (b) Stability of competent cells after long-term storage in liquid nitrogen. Cells were grown at 18C and harvested at an A6oo of 0.60. Each point indicates the average value of duplicate experi- ments.

(g) Effect of plasmid size The influence of plasmid DNA size on the transfor-

mation frequency of our competent cells was assessed by tra~sfecting plasmid DNA whose size ranged from 4.4-17.6 kb. As shown in Fig. 6, the transformation fre- quency is almost e,,ual for plasmids 4.4-7.3 kb in size on a molar basis. When plasmids with sizes of 10.3 kb, 13.2 kb and 17.6 kb were used, relative transformation frequency decreased to 46~o, 26% and 15% of that of pBR322 (4.4 kb). Thus, plasmids up to 10 kb long are able to trans-

(%)

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Plasmid size (kb) Fig. 6. Effect of plasmid size on transformation frequency. The plasmid DNAs with the size of 4.4 kb (pBR322), 6.0 kb, 7.3 kb, 10.3 kb, 13.2 kb and 17.6 kb were transfected to the same lot of competent cell. The ordinate denotes the relative value of transformation frequency (per molecule of each plasmid DNA) when the value ofpBR322 was taken as 100%. Average values of duplicate measurements were plotted.

form this competent cell within a twofold difference in frequency. Competent cells with this size limitation are generally considered sufficient for constructing eDNA libraries. Since the majority of mRNA ranges from 1-6 kb, plasmid vectors of up to 4 kb such as the Okayama-Berg vector can accomodate 6kb cDNA inse~*.s without jeopardizing transformation frequency. Hence, use of com- petent cells prepared by this method is expected to yield a cDNA library representing the original population of m R N A

(h) Comparison with electro-transformation Transformation of E. coli with high-voltage electropo-

ration has been described by Dower et al. (1988). They claimed that it yielded 109-101 transformants/#g plasmid DNA. We tested this method for comparison. It gave a transformation frequency of 1-2 x 109 efu/#g of pBR322 (DNA in TE), a value comparable to the frequency of our chemical method. The major disadvantage of the electro- transformation method, however, is that transformation frequency is greatly influenced by salts and other chemicals generally contained in a ligated DNA solution (Table I). Therefore, their removal prior to electroporation is essential for obtaining the best results. This step is cumbersome, and tends to lose DNA during removal of the interfering chemi- cals, making eleetroporation less desirable for such pur- poses as construction of high-complexity eDNA libraries.

(i) Competence of other Escherichia coil strains The applicability of this method to other E. coli strains

was assessed by testing HBI01 (Boyer and Rouland-

TABLE I

Effect of salt concentrations on transformation frequency of competent cells prepared by our chemical method (SEM) and electroporation

SEM a Electroporation b Transformation (cfu/#g of pBR322 DNA) c frequency

TE a 1.6 109 1.1 109 TE/3 e 1.5 109

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Okayama, H. and Berg, P.: High-efficiency cloning of full-length eDNA.

Mol. Cell. Biol. 2 (1982) 161-170. Okayama, H. and Berg, P.: A cDNA cloning vector that permits expres-

sion of eDNA inserts in mammalian cells. Mol. Cell. Biol. 3 (1983)

280-289. Okayama, H., Kawaichi, M., Brownstein, M., Lee, F., Yokota, T. and

Arai, K.: High-efficiency cloning of full-length eDNA; construction

and screening of eDNA expression libraries for mammalian cells. Methods Enzymol. 154 (1987) 3-28.

Taketo, A.: Sensitivity of Escherichia coli to viral nucleic acid, V. Com- petence of calcium-treated cells. J. Biochem. 72 (1972) 973-979.

Taketo, A. and Kuno, S.: Sensitivity of Escherichia coli to viral nucleic acid, VI. Further studies on Ca: + -induced competence. J. B::~ochem. 75 (1974) 59-67.