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Plasmid DNA transformation in Escherichia Coli: effect of heat shock temperature, duration, and Cold Incubation of Ca[Cl.sub.2] treated cells.

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 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 Ca 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 ([Ca.sup.2+], [Mn.sup.2+], [Sr.sup.2+], [Ba.sup.2+], [Mg.sup.2+], [Na.sup.+] and [Rb.sup.+]), [Ca.sup.2+] (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 [10.sup.5]-[10.sup.7] transformants/[micro]g 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 DH5[alpha] -T1[R] (genotype: [F.sup.-][Phi]80lacZ[Delta]M15 [Delta](lacZYA-argF)U169 rec A1 endA1 hsdR17([r.sub.k.sup.-], [m.sub.k.sup.+]) phoA supE44 thi-1 gyr A96 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 [micro]g/ml. super coiled pUC19 plasmid DNA (2686bp long) was used as a transforming DNA.

Transformation procedure

The Ca[Cl.sub.2] treated 50[micro]l aliquots of competent cells stored at -80[degrees]C were thawed at ice for 30 min. Five [micro]l 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 [micro]l of SOC media to each vial. The vials were finally incubated at 37[degrees]C for 1 hr at 225 rpm in a shaking incubator. The cultures were appropriately diluted in LB medium and 50-100[micro]l of each culture was plated in triplicates on ampicillin containing media plates. These plates were incubated at 37[degrees]C overnight and the resulting transformant colonies were scored and analyzed.

Calculation of transformation efficiency

The transformation efficiency (transformants/[micro]g DNA) was calculated as follows:
 x 300[micro]l total
 # of colonies x [10.sup.6] pg volume
50 pg transformed [micro]g [micro]l volume plated
 DNA

 # of colonies x dilution factor = transformants
50 pg transformed [micro]g plasmid DNA
 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 DH5[alpha] -T1[R] cells, Ca[Cl.sub.2] treated cells of this strain were incubated at 42[degrees]C in a water bath for five different time points i.e. 1, 30, 60, 90 and 120 seconds, individually. The transformation efficiency (x[10.sup.8]) observed after these treatments was 1.21 [+ or -] 0.23; 2.98 [+ or -] 0.16; 2.62 [+ or -] 0.22; 1.92 [+ or -] 0.23 and 0.96 [+ or -] 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 DH5a -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 OMITTED]

Effect of heat shock temperature on transformation:

Four different temperatures i.e. 32[degrees]C, 37[degrees]C, 42[degrees]C and 47[degrees]C were tested to determine the optimum temperature resulting in higher efficiency of transformation of E. coli DH5[alpha]. A fixed incubation time of 30 seconds was used for each treatment. As shown in Fig. 2, we observed transformation efficiency (x[10.sup.8]) of 2.07 [+ or -] 0.08, 2.4 [+ or -] 0.13, 3.31 [+ or -] 0.44 and 1.32 [+ or -] 0.17 for 32[degrees]C, 37[degrees]C, 42[degrees]C and 47[degrees]C heat shock treatments, respectively. These results show that a temperature of 42[degrees]C is optimum to obtain maximum transformation in CaCl2 treated E. coli DH5a cells. Transformation efficiency is significantly reduced at an elevated temperature of 47[degrees]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[degrees]C is optimum temperature for those genes to be expressed at optimal levels. The temperatures higher than 42[degrees]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 OMITTED]

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 in Fig. 3A. Our results indicate that the transformation takes place even before heat shock treatment is given to the Ca[Cl.sub.2] 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 [Mg.sup.2+] and [Ca.sup.2+] 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 OMITTED]

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[degrees]C for an hour in shaking incubator. This high temperature of 37[degrees]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 [Ca.sup.2+] 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 Ca[Cl.sub.2] treated cells to room temperature.

In conclusion, our results suggest that a heat shock pulse of 30 seconds at 42[degrees]C followed by a 10 min ice incubation step are ideal parameters to obtain maximum efficiency of transformation in DH[alpha] -T1[R] 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.+

References

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Mahipal Singh *, Arpita Yadav, Xiaoling Ma and Eugene Amoah

Animal Science Division, Fort Valley State University, Fort Valley, GA 31030, USA

* Corresponding author: Email: singhm@fvsu.edu, mahipal55@hotmail.com
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Author:Singh, Mahipal; Yadav, Arpita; Ma, Xiaoling; Amoah, Eugene
Publication:International Journal of Biotechnology & Biochemistry
Date:Oct 1, 2010
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