Optimization of RK2-based gene introduction system for Bacillus subtilis
| Autor: | Masakazu Kataoka, Hirotada Mori, Mitsuhiro Itaya, Takahiro Yokoi |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0303 health sciences biology 030306 microbiology Chemistry Mutant Bacillus subtilis biology.organism_classification medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology Microbiology 03 medical and health sciences Transformation (genetics) Plasmid Biochemistry 010608 biotechnology medicine Homologous recombination Gene Escherichia coli Bacteria |
| Zdroj: | The Journal of General and Applied Microbiology. 65:265-272 |
| ISSN: | 1349-8037 0022-1260 |
| Popis: | 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 crossover recombinants. The optimized conjugation method described here allowed facility and efficient gene introduction into B. subtilis from E. coli. |
| Databáze: | OpenAIRE |
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