Author(s):
Mendes, Gabriel ; Vieira, Pedro S. ; Lanceros-Méndez, S. ; Kluskens, Leon ; Mota, M.
Date: 2015
Persistent ID: http://hdl.handle.net/1822/35361
Origin: RepositóriUM - Universidade do Minho
Project/scholarship:
info:eu-repo/grantAgreement/FCT/5876-PPCDTI/68393/PT
;
info:eu-repo/grantAgreement/FCT/3599-PPCDT/132966/PT;
info:eu-repo/grantAgreement/FCT/COMPETE/132974/PT;
info:eu-repo/grantAgreement/FCT/5876-PPCDTI/126270/PT
;
info:eu-repo/grantAgreement/FCT/SFRH/SFRH%2FBD%2F71661%2F2010/PT;
info:eu-repo/grantAgreement/FCT/COMPETE/126270/PT;
Subject(s): Yoshida effect; Transformation; Sepiolite; E. coli; Science & Technology; Ciências Médicas::Biotecnologia Médica
Description
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.
Portuguese Foundation for Scienceand Technology (FCT) and the European Community fund FEDER,through Program COMPETE, under the scope of the Projects FCOMP-01-0124-FEDER-007025 (PTDC/AMB/68393/2006), PEst-OE/EQB/LA0023/ 2013, PEST-C/FIS/UI607/2013, RECI/BBB-EBI/0179/2012 (FCOMP-01- 0124-FEDER-027462) and the Projects “BioEnv — Biotechnology and Bioengineering for a sustainableworld” and “Matepro—Optimizing Materials and Processes”. NORTE-07-0124-FEDER-000048 was co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER. The authors also acknowledge the fellowship awarded to Gabriel Mendes SFRH/BD/71661/2010 under the scope of the MITPortugal Program.
