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Metabolic Engineering - Volume 11, Issue 6, November 2009, Pages 398–408

Metabolic engineering of Geobacillus thermoglucosidasius for high yield ethanol production

DOI: 10.1016/j.ymben.2009.08.005

2.8. Transformation of G. thermoglucosidasius

Transformation of G. thermoglucosidasius NCIMB 11955 and DL33 was carried out by electroporation using a medium containing 0.5 M mannitol, 0.5 M sorbitol and 10% (V/V) glycerol for the preparation and electroporation of cells grown in TGP broth. Aliquots of electrocompetent cells (60 μL) were transformed with approximately 200–500 ng (NCIMB 11955) or approximately 50 ng (DL33) of plasmid DNA using a Stratagene Electroporator 1000 (10 μF, 600 Ω, 25 kV/cm). Transformed cells were incubated in pre-warmed TGP medium at 52 °C for 1–4 h with shaking to permit recovery of cells. Transformants (103–104 transformants per μg of plasmid DNA) were selected on TGP agar containing 12.5 μg/mL kanamycin at 52 °C, and recovered after 24 h.

Thank you sir for replying. Sir i have tried out this protocol and we have biorad micropulser and given a pulse at 2.5kv using 2mm cuvette and i used the same TGP medium but not able to get colonies. 

Thank You sir.