Transformation of Escherichia coli JM109 using pUC19 by the Yoshida effect

Abstract

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 1.4×10(9) bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30s). Efficiency values up to 4.1×10(4) 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.

Magnesium aminoclays as plasmid delivery agents for non-competent Escherichia coli JM109 transformation

“…The cell optical density used in this assay was 1.0 (OD 600 = 1.0). Contrary to what was found with sepiolite, in which the best spreading time for manual spreading was 30 s (Mendes et al, 2015), the best spreading time for Mg aminoclays was 60 and 90 s. For 60 s the mean efficiency of transformation obtained was 5.9 × 10 3 CFU/μg pUC19. For 90 s, the mean efficiency of transformation reached a value of 7.0 × 10 3 CFU/μg pUC19 and this was the highest transformation efficiency obtained after all experimental adjustments.…”

“…The maximum value of transformation efficiency obtained in this work is lower than the one obtained with the same host and plasmid using sepiolite through the Yoshida effect (transformation by penetration of nano and micro-sized fibers that form permeation intermediates due to friction on a hydrogel), which achieved a transformation efficiency of 4.1 × 10 4 CFU/μg pUC19 and also does not require the usage of competent cells (Mendes et al, 2015). However, Mg aminoclays are a more user friendly and adequate material than sepiolite and other alternatives for being employed as delivery agents as will be further discussed in this section.…”

Clay-induced DNA breaks as a path for genetic diversity, antibiotic resistance, and asbestos carcinogenesis

“…They may be more sensitive to suffering direct (physical) or indirect (chemical) DNA damage and develop into a mesothelioma. Finally, the sepiolite transformation technique gained in popularity in recent years because there is no need to prepare competent cells 9 , 19 , 35 , 51 . In this case, diverse bacteria can be transformed 4 in both stationary and exponentially growing phases.…”

Clay-induced DNA double-strand breaks underlay genetic diversity, antibiotic resistance and could be a molecular basis for asbestos-induced cancer

“…They may be more sensitive to suffer direct (physical) or indirect (chemical) DNA damage and become into a mesothelioma. Finally, sepiolite transformation technique gained some popularity in the last years because there is no need to prepare competence cells [9, 19,42,52]. In that case, diverse bacteria can be transformed [4] in both stationary and exponentially growing phases.…”