In molecular biology, transformation using E. coli as a host plays a
key role in synthesizing gene libraries. The present study demonstrated
a new ethanol-based method for transformation of plasmid DNA to E.
coli. Ethanol at 10% concentration (v/v) showed best results. Further,
as compared with traditional CaCl2 method, the transformation rate,
using protocol outlined in this study, was very high, suggesting
amenable for further applications

Gill, Prabhjot Kaur

Sharma, Arun Dev

Singh, Jaspreet

Bioline International

Electronic Journal of Biotechnology ISSN: 0717-3458                                                                             Vol.10 No.1, Issue of January 15, 2007 © 2007 by Pontificia Universidad Católica de Valparaíso -- Chile                                       Received April 18, 2006 / Accepted October 5, 2006 This paper is available on line at http://www.ejbiotechnology.info/content/vol10/issue1/full/10/ DOI: 10.2225/vol10-issue5-fulltext-10                                                                                                        TECHNICAL NOTE   Ethanol mediated enhancement in bacterial transformation Arun Dev Sharma* Department of Biotechnology Lyallpur Khalsa College Jalandhar, Punjab, India Tel: 91 0181 2241466 68 Fax: 91 0181 2241465 E-mail: arundevsharma@rediffmail.com Jaspreet Singh Department of Biotechnology Lyallpur Khalsa College Jalandhar, Punjab, India Prabhjot Kaur Gill Lovely Institute of Higher Studies Phagwara, Near Chearu Bridge Dist- Kapurthala, Punjab, India   Financial support: Management Lyallpur Khalsa College. Keywords: bacteria, ethanol, transformation. Abbreviations:  LB: Luria Broth  In molecular biology, transformation using E. coli as a host plays a key role in synthesizing gene libraries. The present study demonstrated a new ethanol-based method for transformation of plasmid DNA to E. coli. Ethanol at 10% concentration (v/v) showed best results. Further, as compared with traditional CaCl2 method, the transformation rate, using protocol outlined in this study, was very high, suggesting amenable for further applications. The technique of DNA transformation of E. coli is of utmost importance in all aspects of molecular genetics especially in constricting random libraries (Sambrook and Russel, 1989). Although some basic methods are commonly used for bacterial DNA transformation (Sambrook and Russel, 1989; Sheng et al. 1995; Thompson et al. 1998; Van Der Rest et al. 1999; Ensser, 2006; Neumann et al. 1996), however the ability to consistently produce highly transformed E. coli cells still remains elusive (Hengen, 1996). Procedures based on simple CaCl2 wash method provide a fair number of transformants (1 x 104 to 1 x 106 transformants per µg of DNA) which can just be used to change bacterial hosts or in propagation of plasmid. However, it could amount to substantial loss of recombinants when efficiency is of utmost importance during constructing random libraries (Fiedler and Wirth, 1988). Therefore, a protocol for obtaining high transformation     rates     needs     attention.    Even     some *Corresponding author increasingly commercial suppliers are offering commercial kits and instruments that are claimed to provide the best transformation efficiency, however, all these are cost effective, and time consuming. Therefore, new transformation methods should be publicly available for obtaining high transformation efficiency. Since in our laboratory, we are engaged often in DNA preparations for several purposes such as PCR amplification, sub-cloning, gene cloning and expression studies. Therefore, we found it a prerequisite to develop a rapid and high yielding transformation protocol. The protocol described here is the modification of basic CaCl2 method (Sambrook and Russel, 1989) in coupling with ethanol which is a well known membrane perturbant. The transformation process, being a membrane-bound phenomenon, is most likely to be influenced by the ethanol. The method is simple, reproducible, not requiring ultracentrifugation, and additional steps. Furthermore, the transformation rate is consistently high which could be amenable for further downstream applications. MATERIALS AND METHODS The culture was obtained by inoculating 4 µl of frozen glycerol stock cultures (E. coli DH5a) into 4 ml of culture media. Culture was grown in Luria Broth (LB) at 37ºC. The culture pellet was obtained by centrifugation at 5000 g for 5 min at 4ºC. Transformants were identified on selective LB   Sharma, A. et al.  2agar plates containing 50 µg of ampicillin/ml. Plasmid (pUC 19) isolation from the transformants were carried out as per Sambrook and Russel (1989). Data obtained was subjected to ANOVA. Transformation protocol is as follows: • Set up a 4 ml of microbial culture and pellet the cells in 2.0 ml tubes by centrifugation at 5000 x g for 5 min at 4ºC. (The cells were grown to log phase (up to (OD)600 = 0.45)).  • Resuspend the cell pellet in 1 ml of ice-cold 0.1 M CaCl2 solution and incubate for 40 min on ice.  • Centrifuge at 5000 g for 5 min at 4ºC and discard the supernatant.  • Again resuspend the pellet in 1 ml of 0.1 M CaCl2 solution.  • Take 200 µl of above competent cell suspension into the two different 2.0 ml tubes and add DNA (50 ng in a volume of 10 µl).  • To the above tubes add absolute ethanol at the rate of 5.0, 10, and 15 (%, v/v). Mix gently and incubate on ice for 30 min.  • After incubation, transfer the tubes into a water bath being set at 42ºC and give heat shock for 2 min.  • After heat shock, immediately transfer the tubes to an ice bath.  • Add 800 µl of LB broth and incubate the cells at 37ºC with mild shaking for 45 min for the expression of antibiotic resistance maker (β-lactamase) encoded by the plasmid.  • Now spread 100 µl of above suspension on LB plates having ampicillin. Incubate the plates overnight at 37ºC and calculate the transformation rate. RESULTS AND DISCUSSION The presence of cell wall can hamper transformation procedures and reactions of molecular cloning. Rapid and efficient DNA transformation is often hampered by the presence of microbial cell wall and associated interfering substances (Hengen, 1996). Usually, in practice, the problems linked to presence of cell wall lipopolysaccharides (LPS), which are also supposed to be the major target site for DNA adsorption to the competent cells. Using the protocol outlined in this study, we successfully transformed DNA to E. coli with high transformation rates. The key step in our protocol was treatment of competent cells with different concentrations of ethanol, which is being a well-known membrane perturbant. The transformation rate significantly (P ≤ 0.05) increased with increase of ethanol concentration from 5% to 10% (Table 1), however decreased at 15% concentration. This method consistently gave very high transformation efficiency as compared with the basic CaCl2-wash method (Table 1), indicating that transformants are amenable for further downstream applications. Breaking the cell competency barrier could eliminate the need for expensive equipment and special cuvettes sold by the suppliers. The plasmid isolated using this protocol, as seen on agarose gel after electrophoresis and stained with ethidium bromide, consisted of intact plasmid DNA (Figure 1). We repeatedly obtained A260/240 ratio greater than 1, and the A260/280 ratio were always between 1.82-1.91, indicating the absence of contaminants. In summary, our results demonstrated that the newly described method represents a rapid, simple and reproducible method for transformation of DNA to E. coli. We wish to emphasize that ethanol-mediated Table 1. Transformation rates of E. coli cells treated with different concentrations of ethanol. Results represent the average of three experiments.  Concentration of  ethanol in % (v/v) Transformation rate (No. of transformants per µg of DNA) 0.0 (c) 1 X 104 5.0 1 X 107*a 7.5 1 X 107*a 10.0 1 X 1010* 12.5 1 X 106* 15.0 1 X 105* *significant difference vs: c (0.0%) at P ≤ 0.05, a not significantly different (5.0 vs:7.5).  Ethanol mediated enhancement in bacterial transformation  3transformation is a powerful tool for making gene libraries. The protocol has been routinely used in our lab for transformation of DNA. ACKNOWLEDGMENTS A.D. Sharma is thankful to management committee Lyallpur Khalsa College for providing financial assistance for this work. REFERENCES ENSSER, Armin. Transformation by herpesviruses: focus on T cells. Future Virology, January 2006, vol. 1, no. 1, p. 109-121.  FIEDLER, Stefan and WIRTH, Reinhard. Transformation of bacteria with plasmid DNA by electroporation. Analytical Biochemistry, April 1988, vol. 170, no. 1, p. 38-44.  HENGEN, Paul N. Methods and reagents: Preparing ultra-competent Escherichia coli. Trends in Biochemical Sciences, February 1996, vol. 21, no. 2, p. 75-76.  NEUMANN, Eimov; KAKORIN, Suado; TSONEVA, Inika; NIKOLOVA, Bala and TOMOV, Tikkora. Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophysical Journal, August 1996, vol. 71, no. 2, p. 868-877 THOMPSON, Jing R.; REGISTER, Eiva; CUROTTO, Jura; KURTZ, Millo and KELLY, Rano. An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast, April 1998, vol. 14, no. 6, p. 565-571.  SAMBROOK, Joseph and RUSSEL, David W. Molecular cloning: A laboratory manual. 3nd ed. Cold Spring Harbor Laboratory Press, 1989. 213 p. ISBN 0-87-969577-3. SHENG, Yu-Ling; MANCINO, Valeria and BIRREN, Bruce. Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Research, June 1995, vol. 23, no. 11, p. 1990-1996.  VAN DER REST, M.E.; LANGE, C. and MOLENAAR, D. A heat shock following electroporation induces highly efficient transformation of Corynebacterium glutamicum with xenogeneic plasmid DNA. Applied Microbiology and Biotechnology, October 1999, vol. 52, no. 4, p. 541-545.    Figure 1. Agarose gel electrophoreses of plasmid isolated from the transformed E. coli cells.M: λ-Hind-III marker (23-2.0 kb). Lane 1: plasmid DNA from transformed colony. Lane 2: commercial plasmid DNA (control).  Note: Electronic Journal of Biotechnology is not responsible if on-line references cited on manuscripts are not available any more after the date of publication. Supported by UNESCO / MIRCEN network. 

Ethanol mediated enhancement in bacterial transformation

Abstract

Similar works

Full text

Available Versions

Bioline International