Biolistic transformation of a procaryote, Bacillus megaterium

Abstract

We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar overlay containing 50 ,Ig of kanamycin per ml. Antibiotic-resistant transformants were recovered from the medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully transformed by this method, although transformation efficiency varied among strains. Physical variables of the biolistic process and biological variables associated with the target cells were optimized, yielding >104 transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.

Genetic Transformation of Peach Tissues by Particle Bombardment

“…As the concentration of osmoticum increased, the number of blue cells increased, until it reached a maximum near 0.3 M. At this concentration, the number of blue cells was doubled compared to the control. The beneficial effect of incorporating osmoticum in the bombardment media has also been observed in yeast (Armaleo et al, 1990), E. coli (Smith et al, 1992), Bacillus (Shark et al, 1991), tobacco suspension cells (Russell et al, 1992), and tobacco chloroplasts , although the optimal concentration for these species varies greatly, ranging from 0.25 to 1.75 M. Osmotic adjustment in the bombardment medium is believed to be responsible for stabilization of the cell membranes for better healing of the lesions caused by particle penetration, and reduction of turgor pressure in the target cells, thus reducing cytoplasmic leakage and cell rupture.…”

“…Cell age and physiological stage are known to have an important effect on transformation efficiency. In bacteria and yeast, cells from early log and stationary phase, respectively, are transformed most efficiently (Shark et al, 1991;Smith et al, 1992). For tobacco suspensions, cells bombarded 4 days after subculture yielded the highest rate of transformation (Russell et al, 1992), while cells 6 days or older produced a much lower number of transformants.…”

Study on the electro-transformation conditions of improving transformation efficiency for Bacillus subtilis

“…The transformation efficiency of B. subtilis varies widely among strains. It was reported that addition of sorbitol could enhance the transformation efficiency of Bacillus megaterium (Shark et al 1991). Xue et al (1999) improved the methods of Vehmaanperä (1989) by adding sorbitol and mannitol into the electroporation medium, which made the cell shrink and reduced the tightness of the membrane.…”

Electrosprayed chitosan nanoparticles: facile and efficient approach for bacterial transformation

“…Common methods for transforming bacterial cells with exogenous DNA are chemo-transformation (mostly, treatment with CaCl 2 ) [1] and electroporation [2]. Other transformation methods such as laser irradiation [3], sonoporation [4], biolistic transformations [5] may also be observed in the literature. Nevertheless, these methods are complicated, expensive and damaging to cells.…”