RESUMO
Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.
Assuntos
Fagos Bacilares/genética , Bacillus subtilis/virologia , Sistemas CRISPR-Cas , Fagos Bacilares/fisiologia , Engenharia Genética , Mutagênese , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Risk is an unavoidable "partner" of the life course for single home dwelling elderly, especially in the older age. The primary concerned person is not the only one to make decisions, his/her proxy and the community care professionals are also involved. Crisscrossing the point of view of these three types of actors with the same situation shows that risk is a notion which has to be problematized. Due to the large part of underlying life principles its understanding can't easily be objectified nor easy to reach by consensus. The three case studies presented suggest a dynamic grid of interpretation for an approach of risk concept.
