Ultrasound-mediated DNA transfer for bacteria.
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 DH5alpha and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl(2), temperature of 22 degrees C, plasmid concentration of 0.8 ng/microl, P. putida UWC1 cell concentration of 2.5 x 10(9) CFU (colony forming unit)/ml and reaction volume of 500 microl, 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 DH5alpha 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.
Song
Yizhi
Y
Centre for Ecology & Hydrology, Oxford, OX1 3SR, UK.
Hahn
Thomas
T
Thompson
Ian P
IP
Mason
Timothy J
TJ
Preston
Gail M
GM
Li
Guanghe
G
Paniwnyk
Larysa
L
Huang
Wei E
WE
D001419:Bacteria / Q000235:genetics*; D002122:Calcium Chloride; D002470:Cell Survival; D003227:Conjugation, Genetic; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000008:administration & dosage*; D018274:Electroporation; D004926:Escherichia coli / Q000235:genetics; D018014:Gene Transfer Techniques; D010957:Plasmids / Q000235:genetics; D011551:Pseudomonas fluorescens / Q000235:genetics; D016958:Pseudomonas putida / Q000235:genetics; D013696:Temperature; D013997:Time Factors; D014169:Transformation, Bacterial; D014465:Ultrasonics
D003470:Culture Media; D004269:DNA, Bacterial; D002122:Calcium Chloride