Methods and reagents: Electro-transformation of Escherichia coli with plasmid DNA Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses some reasons for switching to electroporation to transform bacteria with plasmid DNA. For details on how to partake in the newsgroup, see the accompanying box. Electro-transformation is perhaps the best method for introducing recombinant DNA into bacteria. Many different species of bacteria can be transformed by subjecting a mixture of cells and DNA to short, intense pulses of electrical current. For Escherichia coli [1,2] and Salmonella typhimurium [3], efficiencies of up to 1 x 10^10 transformants per ug of supercoiled plasmid DNA are typical, and can be done with as little as 4 pg of DNA. This is a 10-100-fold increase in transformants when compared with cells that are induced chemically to `competency'. The electrical pulses are on the order of 5-20 kV/cm, and are usually produced by exponential-decay waveform, which is generated by the discharge of a capacitor [1]. Although many people's first reaction may be that this is a complicated or difficult procedure, electroporation is very fast and simple. In addition, electro-competent E. coli cells are easy to prepare and may be made in advance and frozen. As in many other chemical transformation procedures, cells are grown at 37 degrees C until early to mid-log phase, which is determined by an optical density reading of approximately 0.35-0.4 at 600 nm. This is generally reached 2.5-3 h after diluting a stationary phase culture 1:50 and placing the flask in a shaking incubator. [See TIBS 19(1994), 426-427 for a discussion of the optimal cell-density for chemical transformation of E. coli]. The cells are collected by centrifugation, washed several times in sterile distilled water and resuspended in ice-cold 10% glycerol (prepared without autoclaving or filter-sterilizing). Afterwards, the cells are concentrated to 1-3 x 10^10 colony-forming units per ml. In a cold, 1.5 ml polypropylene tube, 40 ul of the cell suspension is mixed with 1-2 ul of DNA at a concentration of 10 pg/ul. The mixture of cells and DNA is transferred to a cold electroporation cuvette; it is important to make sure that the liquid is in contact with both electrodes, which may be 0.10-0.40 cm apart. For E. coli it has been found that one electrical pulse is optimal, with a field strength of 16-18 kV/cm and a time constant of 4.5-5.5 ms, depending on the strain to be transformed. Interestingly, when pulse length or the number of pulses is increased, a larger percentage of cells die, but higher transformation frequencies are obtained when the survival rate is between 25 and 50%. Cells are immediately diluted in a recovery medium and allowed to express plasmid-encoded markers before they are plated on selective media. Bacteria should be spread onto dry agar plates with either a glass hockey stick or glass spheres; this cann be done by tipping a few sterile 3-4 mm glass beads onto a plate with the bacteria and rolling them around until the plate is dry, then tipping them out into a beaker of ethanol for re-use. Although electroporation is the most efficient method for introducing DNA into bacteria, it is also the most expensive. So is it worth the money to switch from the CaCl2 method of chemical transformation to electroporation for introducing recombinant plasmids into E. coli? While it is important to get an efficient transformation for some experiments, such as for the construction of genomic libraries, it may not be worth the extra expense for other routine transformations. The efficiency obtained by most other methods for re-introducing a plasmid into a different strain of E. coli or subcloning a restriction fragment into a different vector will not warrant the purchase of such an expensive piece of equipment. To operate at a high transformation level for less cost, some netters build their own personal electroporation units and use them for all their bacterial transformations.[4] One netter has even used electroporation routinely for subcloning experiments and wrote that ligations that have been done within solidified SeaPlaque [R] agarose (FMC, BioProducts, Rockland ME, USA) can also be used for electro-transformation. He found that it is not necessary to dialyze out the electrophoresis buffer components before zapping the cells, and that a 4 ul aliquot of the ligation mix within molten agarose diluted 1:1 according to the protocol of Kalvakolanu and Livingston [5] gives excellent transformation frequencies. The secret life of an electroporation cuvette ********************************************* The major drawback with this technique is the cost. Not only is the electrical unit itself expensive, but special electroporation cuvettes supplied for the apparatus are pricey. For example, cuvettes for the Bio-Rad Gene Pulser [TM] or E. coli Pulser [TM] (Bio-Rad Laboratories, Hercules, CA, USA), which can be equipped with special 0.10, 0.20, or 0.40 cm gapped electrodes, are very expensive since they come in packages of 50 prewashed, presterilized and individually wrapped cuvettes intended for single use. Netters claim that if they are washed and re-used, the cuvettes can last as long as a year. Connie Rickey of Bio-Rad Laboratories (crickey@haley.genetics.bio-rad.com) suggests that caution should be exercised if you plan to re-use cuvettes. Apart from the obvious sterility and cross-contamination risks, lower efficiencies may be obtained owing to pitting of the electrodes. Since a high-voltage pulse induced upon a concentrated bacterial solution might cause the cells in contact with the aluminum surface of the cuvette to bake onto the electrode, and because an obstruction of dead cells has the same electrical effect as a pit on the electrodes, they may become unusable relatively quickly. Electro- transformation efficiencies are highest in a uniform electric field; that is, between two completely conductive parallel electrodes. She feels that if they do become pitted, they should definitely not be used because they could possibly be unsafe for passing high voltages. Some older cuvettes can cause a very loud `pop' and the lid can be blown off when an electrical arc is formed. Any that have visible cracks in the glass should be discarded. Cleaning them with a harsh chemical, such as strong acid or base, or even bleach, might cause the smooth surfaces of the electrodes to get abraded and eventually worn away, which would affect the transformation efficiency adversely. However, opinions vary concerning the life expectancy of cuvettes, and netters feel that they can be re-used many times before the electrodes become corroded or gummed. Cuvettes can simply be washed out with concentrated bleach, rinsed at least six times with distilled water and twice with 95% ethanol, and stored inverted within an exhaust hood or under vacuum. For more careful work, the cuvettes can be filled with 0.25 M hydrochloric acid or 30% nitric acid and allowed to stand for 15 min to 1 h at room temperature on the lab bench, before being rinsed several times, as above. Also, keeping the cuvettes stored within a desiccator will most probably prolong their lab-life since a major problem is deterioration from leftover salts and moisture. Those who have cleaned and re-used the cuvettes in the above fashion say that they have not experienced any carryover of cells or DNA. On your markers *************** An additional consideration is the genetic markers to be `electro-duced' into the cell. Lower transformation frequencies are seen when selection is made for tetracycline resistance, rather than for ampicillin resistance. If bacteria are plated onto media containing tetracycline or a related antibiotic, they cannot recover, no matter how long the cells are allowed to express. Most probably, damage inflicted by the high-voltage shock to the membrane is not repaired properly and the antibiotic is allowed into the cell before it has a chance to recover. The result is a transformation efficiency comparable to that of chemical transformation. Therefore, money spent on an electroporation apparatus for construction of a gene library within a vector encoding tetracycline resistance would be wasted. Using a vector with an ampicillin-resistance marker, on the other hand, usually gives a very high transformation efficiency after electroporation, presumably because the active mechanism for breakdown of ampicillin by beta-lactamase can begin before membrane repair ensues, thus allowing a complete and rapid recovery. [6] Given that many of the more widely used cloning vectors are based on the pUC plasmid series, which encodes ampicillin resistance, electroporation and selection for this marker are the best bet for successful, high efficiency cloning. References [1] Dower, W. J., Miller, J. F. and Ragsdale, C. W. (1988) Nucleic Acids Res. 16, 6127-6145 [2] Taketo, A. (1988) Biochim. Biophys. Acta 949, 318-324 [3] O'Callaghan, D. and Charbit, A. (1990) Mol. Gen. Genet. 223, 156-158 [4] Speyer, J. F. (1990) BioTechniques 8, 28-30 [5] Kalvakolanu, D. V. R., and Livingston, W. H., III, (1991) BioTechniques 10, 176-177 [6] Steele, C., Zhang, S. and Shillitoe, E. J. (1994) BioTechniques 17, 360-365 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/jun95.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1995. Methods and reagents - Electro-transformation of Escherichia coli with plasmid DNA. Trends in Biochemical Sciences 20(6):248-249. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************