Nucleic Acids Research Advance Access published online on September 22, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm710
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Methods Online |
Ultrasound-mediated DNA transfer for bacteria
1Centre for Ecology & Hydrology, Oxford, OX1 3SR, UK, 2Department of Environmental Science and Engineering, Tsinghua University, Beijing, 100084, People's Republic of China, 3Begbroke Directorate, University of Oxford Science Park, Yarnton, Oxford, OX5 1PF, 4Faculty of Health and Life Sciences, Coventry University, Coventry, CV1 5FB and 5Department of Plant Sciences, University of Oxford, Oxford OX1 3RB, UK
*To whom correspondence should be addressed. Tel: +44 (0)114 2225796; Fax: +44 (0)114 2225701; Email: weiehuang{at}yahoo.com, w.huang{at}sheffield.ac.uk
Received June 27, 2007. Revised August 14, 2007. Accepted August 27, 2007.
In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5
and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl2, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 x 109 CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 x 10–6 transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5 and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 x 10–6 and 4.33 ± 0.78 x 10–6 transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.
Present address: Wei E. Huang, Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ, UK.