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

Chan WT, Verma CS, Lane DP, Gan SK - Biosci. Rep. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore 138671.

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.

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

Mentions: For comparison of the four methods on the six strains, both ANOVA and independent t tests were used. ANOVA test was performed to determine the reproducibility of the TrE within each method (Supplementary Table S2A at http://www.bioscirep.org/bsr/033/bsr033e086add.htm), as well as the differences between the four methods (Supplementary Table S2B at http://www.bioscirep.org/bsr/033/bsr033e086add.htm). Independent t test was used for the comparison of pair-wise method comparisons (Supplementary Table S2C at http://www.bioscirep.org/bsr/033/bsr033e086add.htm), media (Figure 2), heat-shock incubation times (Figure 3), and four-fold concentration and neat (Figure 4). All statistical analysis was performed using SPSS ver. 17 (IBM) at a 95% confidence interval.


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

Chan WT, Verma CS, Lane DP, Gan SK - Biosci. Rep. (2013)

Comparison of the use of LB or SOB as growth mediaBar 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3860579&req=5

Figure 2: Comparison of the use of LB or SOB as growth mediaBar 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.
Mentions: For comparison of the four methods on the six strains, both ANOVA and independent t tests were used. ANOVA test was performed to determine the reproducibility of the TrE within each method (Supplementary Table S2A at http://www.bioscirep.org/bsr/033/bsr033e086add.htm), as well as the differences between the four methods (Supplementary Table S2B at http://www.bioscirep.org/bsr/033/bsr033e086add.htm). Independent t test was used for the comparison of pair-wise method comparisons (Supplementary Table S2C at http://www.bioscirep.org/bsr/033/bsr033e086add.htm), media (Figure 2), heat-shock incubation times (Figure 3), and four-fold concentration and neat (Figure 4). All statistical analysis was performed using SPSS ver. 17 (IBM) at a 95% confidence interval.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: *Bioinformatics Institute, Agency for Science, Technology, and Research (A*STAR), Singapore 138671.

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.

Show MeSH
Related in: MedlinePlus