A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

Abstract

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl2-CaCl2, CaCl2 and Hanahan's methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan's method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl2 method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria-Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109's competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.

Figure 1. Comparison of the four standardized chemical methods on six strains of E. coli

Bar chart showing the means and standard errors of the TrE obtained from the six strains of E. coli produced using the four different methods. Controls were performed by transforming competent bacteria with water, which did not yield any transformants.

Figure 2. Comparison of the use of LB or SOB as growth media

Bar chart showing the means, standard errors and t tests of TrE obtained from both CaCl₂ and Hanahan methods of chemical induction for the respective strains. (Top panel) Bar chart representing the three strains that responded best to Hanahan's method. (Bottom panel) Bar chart representing the three strains that responded best to CaCl₂ method. * denotes that P<0.05; ** denotes that P<0.001 for the t tests comparing the means. Refer to Supplementary Table S2 at http://www.bioscirep.org/bsr/033/bsr033e086add.htm for detailed statistical analysis. The t test showed that there was no significant difference between the use of SOB or LB media for DH5α, SCS110, TOP10 and BL21 strains despite higher average TrE for SOB media.

Figure 3. Comparison of the 45 and 90 s heat shock

Bar chart showing the means, standard error and t test results of 45 and 90 s incubation times performed across all strains of E. coli made using the optimized method with SOC. The absence of significance differences indicate that the 45 or 90 s heat-pulse yielded similar transformation efficiencies across all strains tested.

Figure 4. Comparison of four-fold concentration with the default final resuspension method in the optimized methods across the six E. coli strains

Bar chart showing the means, standard errors and t test results of the TrE obtained from optimally induced competent bacteria that were concentrated four-fold with that of default aliquots.* denotes that P<0.05; ** denotes that P<0.001 for the t tests comparing the means.