Isotachophoresis for rapid transformation of Escherichia coli.
Electrophoresis
; 43(4): 543-547, 2022 02.
Article
em En
| MEDLINE
| ID: mdl-34837243
ABSTRACT
A frequent limitation of electroporation (EP) and chemical transformation (CT) are the need of tedious and time-consuming procedures for inducing transformation competence, the substantial number of cells required, and the low transformation yields typically achieved. Here, we show a new and rapid electrokinetic method for transformation of small number of noncompetent Escherichia coli TOP10 cells (2-3 × 105 ) at room temperature. Escherichia coli TOP10 cells and plasmid DNA are sequentially injected into a 50 µm ID capillary and focused into 11.5 nL by isotachophoresis (ITP) induced by application of high DC voltage (-16 kV). Through ITP, a large excess of plasmid DNA is brought in contact with the cell surface, with the contact time adjusted by application of a counter-pressure (1.3 psi) opposing the ITP movement. The transformation rate was more than 1000-fold higher compared to EP and CT at survival rates greater than 60%.
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MEDLINE
Assunto principal:
Isotacoforese
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article