Transformation of Escherichia coli JM109 using pUC19 by the Yoshida effect

• Autor(a) principal: Mendes, Gabriel
• Data de Publicação: 2015
• Outros Autores: Vieira, Pedro S., Lanceros-Méndez, S., Kluskens, Leon, Mota, M.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/1822/35361
• topic: Yoshida effect; Transformation; Sepiolite; E. coli; Ciências Médicas::Biotecnologia Médica; Science & Technology

Transformation of non-competent Escherichia coli JM109 was accomplished using pUC19 as donor plasmid and sepiolite as the acicular material to promote cell piercing via application of friction with a polystyrene stick or a magnetic bar on the surface of a hydrogel containing agar. An automatic spreading setup was built with a conventional stirring plate and compared to manual spreading. Several parameters were optimized, namely, the agar content of the hydrogel (2%), concentration of cells (OD=1.3 corresponding to 1.4x109 bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30 sec). Efficiency values up to 4.1 x 104 CFU/µg pUC19 were obtained. The method proved to be suitable for a rapid and low cost transformation of non-competent E. coli JM109, where higher values of efficiency do not need to be attained.