Genetic transformation and transfection ofBacillus subtilis spheroplasts

Tichý, P.; Rytíř, V.; Kohoutová, M.

doi:10.1007/bf02874223

Cited by 8 publications

(2 citation statements)

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“…Despite these favorable characteristics, genetic manipulation systems, that would include transformation, have been developed only for few well-characterized laboratory strains, such as B. subtilis Marburg 168 and PY79. The natural transformation method (Zhang et al 2014) polyethylene glycol-mediated protoplast transformation (Tichý et al 1968), and electroporation method (Masson et al 1989) have been utilized to allow DNA transfer into B. subtilis, but they are not as easy as in E. coli. In addition, these three methods have DNA size limitation due to shear stress and DNA passage during transformation.…”

Section: Introductionmentioning

confidence: 99%

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Yokoi

Itaya

Mori

et al. 2019

J. Gen. Appl. Microbiol.

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The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double

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Section: Introductionmentioning

confidence: 99%

Optimization of RK2-based gene introduction system for <i>Bacillus subtilis</i>

Yokoi

Itaya

Mori

et al. 2019

J. Gen. Appl. Microbiol.

View full text Add to dashboard Cite

show abstract

“…Chargaff et al (1957) did report the transformation of coll spheroplasts; however, the frequency was very low, probably due to the low frequency of reversion to walled forms and the presence of DNases from lysed cells. Hirokawa and Ikeda (1966) and Tichy et al (1968) reported efficient transformation of B. subtilis protoplasts. However, these two studies may have involved "quasi spheroplasts" which Tichy and Landman (1969) found to be highly transformable; these investigators found true lysozyme-induced protoplasts to be nontransformable.…”

Section: +mentioning

confidence: 99%