Plasmid DNA Transformation in Escherichia Coli

International Journal of Biotechnology and Biochemistry ISSN 0973-2691 Volume 6 Number 4 (2010) pp. 561–568 © Research India Publications http://www.ripublication.com/ijbb.htm

Plasmid DNA Transformation in Escherichia Coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation of CaCl2 Treated Cells Mahipal Singh*, Arpita Yadav, Xiaoling Ma and Eugene Amoah Animal Science Division, Fort Valley State University, Fort Valley, GA 31030, USA *Corresponding author: Email: [email protected], [email protected]

Abstract Various parameters of standard CaCl2/heat shock method on transformation of Escherichia coli strain DH5α -T1R with plasmid pUC19 were optimized. Of the four different heat shock temperatures (32°C, 37°C, 42°C and 47°C) studied, 42°C treatment exhibited maximum efficiency of transformation as revealed by ampicillin-resistant colonies appearing on LB Agar ampicillin plates. Of the five different heat shock exposure times, a pulse of 30 second duration combined with 42°C heat shock temperature exhibited maximum efficiency. It was observed that although transformation of CaCl2 treated cells occurs even before heat shock treatment, the efficiency was ~15 fold higher after heat shock. When the cells were further incubated on ice (after heat shock) for 10 min, the transformation efficiency increased by 24 fold compared to no heat shock and 1.6 fold compared to heat shock treatment. There was a marginal decrease in transformation when cells were incubated at room temperature instead of ice after heat shock. These results suggest that a heat shock pulse of 30 sec at 42°C followed by a 10 min ice incubation step are ideal parameters to obtain maximum transformation efficiency in DH5α T1R strain. Results also suggest that post heat shock cold incubation step is also an important factor and enhances transformation of E. coli significantly. Key words: transformation, E. coli, plasmid, CaCl2, competent cells, heat shock.

Introduction The ability to introduce plasmid DNA molecules into the cells has been of central importance to the development of molecular biology. Several methods have been

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reported in the literature to introduce plasmid DNA into the cells. These methods include chemical treatment (1, 2), electroporation (3, 4), use of biolistic gun (5), polyethylene glycol (6), ultrasound (7), microwave (8) and, hydrogel (9). However, the chemical methods have attained much attention in most of the laboratories, due to their accessibility and cost effectiveness. The physiological state of cells that enables them to bind and take up high molecular weight exogenous DNA is called “competence”. Uptake of free DNA by Escherichia coli cells which have become competent by treatment of chemicals providing Ca2+ ions followed by a heat shock pulse was first reported by Mandel and Higa (10). Subsequently, several modifications of this method became available for transformation of E. coli with plasmid DNA (1, 11-13). Among many cations tested (Ca2+, Mn2+, Sr2+, Ba2+, Mg2+, Na+ and Rb+), Ca2+ (100-200 mM range) provided comparatively better transformation of E. coli (14). Physiological conditions for optimum transformation, however, vary from strain to strain, their genetic background, and type of transforming DNA (14). Transformation frequencies obtained using these methods range approximately 105-107 transformants/µg of DNA. The frequencies are still about tenfold lower when a DNA ligation reaction mixture is used as input DNA, resulting in lower recombinant clones per plate. Although for cloning and sub-cloning purposes high efficiency of transformation is not critical, applications such as construction of genomic and cDNA libraries require a very high efficiency of transformation, in order to have proper representation of low copy number sequences. It is, therefore, important to optimize and improve the frequency of transformation of the desired host strain to achieve cloning of low copy number DNA molecules.

Material and Methods: Bacterial Strain, culture media and plasmid DNA used Escherichia coli strain DH5α -T1R (genotype: F-Φ80lacZΔM15 Δ(lacZYAargF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 thi-1 gyrA96 relA1 tonA) was used in this study (Invitrogen Inc.). Luria-Bertani (LB) medium (Sigma Aldrich Inc) was routinely used to culture E. coli. For making plates medium was solidified with 1.6% agar. Antibiotic plates for selection of transformants contained ampicillin (Sigma Aldrich Inc.) at a final concentration of 100 µg/ml. super coiled pUC19 plasmid DNA (2686bp long) was used as a transforming DNA. Transformation procedure The CaCl2 treated 50µl aliquots of competent cells stored at -80°C were thawed at ice for 30 min. Five µl of pUC19 plasmid containing a total of 50pg DNA was directly pipetted over competent cells. These cells were mixed gently by tapping 4-5 times, incubated on ice for 30 min, which was followed by a heat shock treatment. After treatment the cells were routinely incubated at ice for 2 min followed by addition of 250 µl of SOC media to each vial. The vials were finally incubated at 37°C for 1 hr at 225 rpm in a shaking incubator. The cultures were appropriately diluted in LB medium and 50-100µl of each culture was plated in triplicates on ampicillin

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containing media plates. These plates were incubated at 37°C overnight and the resulting transformant colonies were scored and analyzed. Calculation of transformation efficiency The transformation efficiency (transformants/µg DNA) was calculated as follows: # of colonies 50 pg transformed DNA

x 106 pg µg

x 300µl total volume µl volume plated

x dilution factor =

transformants µg plasmid DNA

Results and discussion Effect of duration of heat shock treatment on plasmid transformation Literature shows various times of heat shock treatment ranging from 30 sec to 2 min to obtain transformation of E. coli. There is no consistent report as to how much heat shock time results in maximum transformation efficiency. To determine the optimum time for heat shock treatment for DH5α -T1R cells, CaCl2 treated cells of this strain were incubated at 42°C in a water bath for five different time points i.e. 1, 30, 60, 90 and 120 seconds, individually. The transformation efficiency (x108) observed after these treatments was 1.21±0.23; 2.98±0.16; 2.62±0.22; 1.92±0.23 and 0.96±0.17 for 1, 30, 60, 90 and 120 seconds, respectively (Fig.1). These results indicate that 30 seconds duration is optimum for obtaining maximum transformation efficiency of DH5α -T1R strain with pUC19 plasmid DNA. Longer heat shock treatments significantly reduced the transformation efficiency. It is known that viability of cells decreases with longer exposures at high temperatures and perhaps this might have contributed to the reduction in transformation efficiency observed.

Figure 1: Effect of heat shock incubation time on transformation: Cells were treated for various times at 42°C. Data represent mean ± SD of three repetitions. Each treatment was plated in triplicate.

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Effect of heat shock temperature on transformation: Four different temperatures i.e. 32°C, 37°C, 42°C and 47°C were tested to determine the optimum temperature resulting in higher efficiency of transformation of E. coli DH5α. A fixed incubation time of 30 seconds was used for each treatment. As shown in Fig. 2, we observed transformation efficiency (x108) of 2.07±0.08, 2.4±0.13, 3.31±0.44 and 1.32±0.17 for 32°C, 37°C, 42°C and 47°C heat shock treatments, respectively. These results show that a temperature of 42°C is optimum to obtain maximum transformation in CaCl2 treated E. coli DH5α cells. Transformation efficiency is significantly reduced at an elevated temperature of 47°C. It has been shown that heat shock induces certain genes to express heat shock proteins (HSPs) which help the cells to survive at elevated temperatures, and thus higher transformation efficiency. It may be possible that 42°C is optimum temperature for those genes to be expressed at optimal levels. The temperatures higher than 42°C may be detrimental to the cell survival and thus decrease in transformation efficiency. It may also be possible that incubation at a lower temperature for longer period of time may achieve same results.

Figure 2: Effect of heat shock temperature on transformation: Cells were incubated for 30 sec at four different temperatures individually. Data represent mean ± SD of two repetitions. Each treatment was plated in triplicate.

Effect of cold incubation of heat shock treated cells on transformation: How the transformation efficiency is affected by cold incubation of the bacterial cells after heat shock treatment is not clearly defined. To test the effect of cold incubation (after heat shock) on transformation of E. coli, an experiment was designed as shown


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in Fig. 3A. Our results indicate that the transformation takes place even before heat shock treatment is given to the CaCl2 treated cells (Fig. 3, treatment 1). However, the efficiency of transformation is approximately 15 fold lower as compared to heat shock (Fig. 3B, treatment 1 vs. 2). These results support the earlier observations that heat shock significantly enhances the efficiency of transformation. However, the mechanism of heat shock induced DNA uptake in CaCl2 treated cells is still not precisely known. It has been suggested that bivalent cations such as Mg2+ and Ca2+ play a significant role in interaction of DNA with membrane phospholipids. These cations are bound to phospholipids and provide positive charge to them. Negatively charged DNA tends to attach to the lipid molecules through mediation of bivalent cations (15). Sudden shock by heat alters the membrane and helps DNA internalization at a quicker rate perhaps by cell surface invagination carrying membrane bound DNA into the cells. These assumptions are supported by earlier observations that E coli from which lipopolysaccharides (LPS) have leached out show high efficiency of transformation when transformed with plasmid-LPS complexes rather than plasmid DNA alone (16, 17).

Figure 3: Effect of cold incubation on transformation after heat shock treatment of the cells: Panel-A depicts the experimental design while panel-B shows transformation efficiency after corresponding treatments. Numbers 1, 2, 3 and 4 are four independent treatments as shown in panel A. Tube number 1 never received heat

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shock. Tube number 2 was provided with SOC media immediately after heat shock. Tube number 3 was incubated at room temperature (RT) for 10 min after heat shock treatment before receiving SOC media. Tube number 4 was incubated at ice for 10 min before receiving SOC media. 0°C indicates ice incubation step. Data represent mean ± SD of 3 repetitions. Each treatment was plated in triplicate. Statistical significance between groups was performed by Student unpaired t-test using GraphPad software. Probability values are shown in the graph. As seen in the graph the values are highly statistically significant at the levels < 0.0002 except for one group (treatment 2 vs. 3) which is insignificant.

Interestingly, incubation of the cells at ice after heat shock step additionally increases transformation efficiency by ~1.6 fold (Fig. 3B, treatment 2 vs. 4). When four different cold incubation temperatures (1, 15, 30 and 60 min) post heat shock were tested, 15 min cold incubation showed maximum efficiency of transformation beyond which there was no appreciable increase (data not shown). During the heat shock period the motion of tiny plasmid DNA molecules in the competent cell mixture is likely to increase. It is possible that post heat shock ice incubation step reduces thermal motion of plasmid DNA molecules and thus promote further binding of left-over (plasmid DNA not taken up by cells during heat shock) DNA to cell surface. These additional DNA molecules may be further taken up by cells while they are incubated at 37°C for an hour in shaking incubator. This high temperature of 37°C may serve as a second heat shock step and thus enhances the overall transformation efficiency of E. coli. It has been shown that conversion of DNA into a DNase resistant form occurs during ice incubation after heat shock step (1). It is possible that DNase resistant form of DNA ensured its survival inside the cells and thus resulted in higher transformation efficiency. It seems that the competence induction and DNA uptake are two separate stages and that the heat shock of the Ca2+ treated cells perhaps play an important role in DNA uptake. There seems to be a slight reduction in transformation when the cells are incubated at room temperature rather than ice post heat shock treatment (Fig. 3B treatment 3 vs. 2). Perhaps it is due to inability of some cells to revive after exposure of very fragile CaCl2 treated cells to room temperature. In conclusion, our results suggest that a heat shock pulse of 30 seconds at 42°C followed by a 10 min ice incubation step are ideal parameters to obtain maximum efficiency of transformation in DH5α -T1R strain with pUC19 plasmid DNA. This study also suggests that post heat shock cold incubation step is also an important factor and enhances transformation frequency significantly.

Acknowledgements Ms. Ting He is acknowledged for help with media preparations.+


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References [1] [2] [3] [4] [5]

[6] [7]

[8] [9] [10] [11] [12]

[13]

[14] [15]

[16]

Bergmans HE, van Die IM, Hoekstra WP. Transformation in escherichia coli: Stages in the process. J Bacteriol. 1981 May;146(2):564-70. Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. 1970. Biotechnology. 1992;24:198-201. Dower WJ. Electroporation of bacteria: A general approach to genetic transformation. Genet Eng (N Y). 1990;12:275-95. Drury L. Transformation of bacteria by electroporation. Methods Mol Biol. 1994;31:1-8. Smith FD, Harpending PR, Sanford JC. Biolistic transformation of prokaryotes: Factors that affect biolistic transformation of very small cells. J Gen Microbiol. 1992 Jan;138(1):239-48. Kurien BT, Scofield RH. Polyethylene glycol-mediated bacterial colony transformation. BioTechniques. 1995 Jun;18(6):1023-6. Song Y, Hahn T, Thompson IP, Mason TJ, Preston GM, Li G, et al. Ultrasound-mediated DNA transfer for bacteria. Nucleic Acids Res. 2007;35(19):e129. Fregel R, Rodriguez V, Cabrera VM. Microwave improved escherichia coli transformation. Lett Appl Microbiol. 2008 Apr;46(4):498-9. Yoshida N, Sato M. Plasmid uptake by bacteria: A comparison of methods and efficiencies. Appl Microbiol Biotechnol. 2009 Jul;83(5):791-8. Mandel M, Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159-62. Dagert M, Ehrlich SD. Prolonged incubation in calcium chloride improves the competence of escherichia coli cells. Gene. 1979 May;6(1):23-8. Huff JP, Grant BJ, Penning CA, Sullivan KF. Optimization of routine transformation of escherichia coli with plasmid DNA. BioTechniques. 1990 Nov;9(5):570,2, 574, 576-7. Nakata Y, Tang X, Yokoyama KK. Preparation of competent cells for highefficiency plasmid transformation of escherichia coli. Methods Mol Biol. 1997;69:129-37. Tsai SP, Hartin RJ, Ryu J. Transformation in restriction-deficient salmonella typhimurium LT2. J Gen Microbiol. 1989 Sep;135(9):2561-7. Sato Y, Kumazawa N, Yoshikawa K, Kurusu Y. Transformation of escherichia coli mediated by natural phospholipids. Biosci Biotechnol Biochem. 2005 Jan;69(1):235-7. Panja S, Aich P, Jana B, Basu T. Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2mediated transformation process. Biomacromolecules. 2008 Sep;9(9):2501-9.

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Mahipal Singh et al

[17]

Panja S, Aich P, Jana B, Basu T. How does plasmid DNA penetrate cell membranes in artificial transformation process of escherichia coli? Mol Membr Biol. 2008 Aug;25(5):411-22.