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.
Highlights
Bacterial transformation, the process whereby bacteria are able to take up foreign DNA was first demonstrated by Griffith for Streptococcus pneumonia [1], and is routinely used in laboratories
Reported to occur naturally in bacteria such as Bacillus subtilis [2], such phenomenon is generally uncommon in Escherichia coli, which require induction by artificial methods, such as those first demonstrated by Mandel and Higa [3]
This chemical method involved treating the bacteria with bacteriophage λ DNA in the presence of Ca2 + ions, followed by a brief heat shock [4]
Summary
The process whereby bacteria are able to take up foreign DNA was first demonstrated by Griffith for Streptococcus pneumonia [1], and is routinely used in laboratories. Reported to occur naturally in bacteria such as Bacillus subtilis [2], such phenomenon is generally uncommon in Escherichia coli, which require induction by artificial methods, such as those first demonstrated by Mandel and Higa [3] This chemical method involved treating the bacteria with bacteriophage λ DNA in the presence of Ca2 + ions, followed by a brief heat shock [4]. It was discovered that only 1–2 % of the transformants survived in this method [6], making it inefficient for routine DNA manipulation Later, modifications such as the use of multiple heat-shock cycles [7] and variations of chemicals were tested. These resulted in several optimized versions of the original CaCl2 method [8,9] that typically yield ‘TrE’ (transformation efficiencies) of 5 × 106–2 × 107 colonies forming units per microgram (cfu/μg) of DNA
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