Isotachophoresis for rapid transformation of Escherichia coli |
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| Authors: | Monica N. Alves Yi Heng Nai Shane M. Powell Mirek Macka Michael C. Breadmore |
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| Affiliation: | 1. Australian Centre for Research on Separation Science (ACROSS), University of Tasmania, Hobart, Australia;2. Centre for Regional and Rural Futures (CeRRF), Faculty of Science, Engineering and Built Environment, Deakin University, Geelong, Victoria, Australia;3. Tasman Institute of Agriculture, University of Tasmania, Hobart, Australia |
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| 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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| Keywords: | Bacteria CE Isotachophoresis Plasmid Transformation |
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