DH5-Alpha Cell explained

DH5-Alpha Cells are E. coli cells engineered by American biologist Douglas Hanahan to maximize transformation efficiency. They are defined by three^[1] mutations: recA1, endA1 which help plasmid insertion and lacZΔM15 which enables blue white screening. The cells are competent and often used with calcium chloride transformation to insert the desired plasmid. A study of four transformation methods and six bacteria strains showed that the most efficient one was the DH5 strain with the Hanahan method.^[2]

Mutations

• The recA1 mutation is a single point mutation that replaces glycine 160 of the recA polypeptide with an aspartic acid residue^[3] in order to disable the activity of the recombinases and inactivate homologous recombination.
• The endA1 mutation inactivates an intracellular endonuclease to prevent it from degrading the inserted plasmid.^[4]

Notes and References

1. Web site: Wertz J . Strain - DH5α. The Coli Genetic Stock Center (CGSC) . Yale University . 2017-05-23.
2. Chan WT, Verma CS, Lane DP, Gan SK . A comparison and optimization of methods and factors affecting the transformation of Escherichia coli . Bioscience Reports . 33 . 6 . December 2013 . 24229075 . 3860579 . 10.1042/BSR20130098 .
3. Bryant FR . Construction of a recombinase-deficient mutant recA protein that retains single-stranded DNA-dependent ATPase activity . The Journal of Biological Chemistry . 263 . 18 . 8716–8723 . June 1988 . 2967815 . 10.1016/S0021-9258(18)68364-4 . free .
4. Taylor RG, Walker DC, McInnes RR . E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing . Nucleic Acids Research . 21 . 7 . 1677–1678 . April 1993 . 8479929 . 309390 . 10.1093/nar/21.7.1677 .