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How to transform a recalcitrant Paenibacillus strain: From culture medium to restriction barrier
Evelise Bach1, Gabriela de Carvalho Fernandes1, Luciane Maria Pereira Passaglia1
1Laboratório de Microbiologia Agrícola, Departamento de Genética, Instituto de Biociências (IB), Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil.
Abstract:
Paenibacillus riograndensis SBR5T is a plant growth-promoting bacterium isolated from the wheat rhizosphere. Its recalcitrance to genetic manipulation is a major bottleneck for molecular studies, as has been reported for other Paenibacillus environmental isolates. An efficient electroporation protocol was established by evaluating diverse parameters and optimizing the culture medium, culture growth phase, electroporation solution, recovery medium, DNA input, and electric field strength. Efficiencies of approximately 2.8×104transformantsμg-1 of plasmid DNA were obtained. The optimized protocol was tested with other Paenibacillus species, and the relevance of bypassing the restriction DNA defense system to transform Paenibacillus was highlighted. This protocol is the tool needed to deepen molecular studies with this strain and will aid in the manipulation of other new environmental isolates that also exhibit recalcitrant transformation difficulties.
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