Fig. 4

Silencing of the ncRNA RNase P in L. biflexa. (A) L. biflexa cells containing plasmids pMaOri.dCas9 alone, or with sgRNA cassettes for pairing to the sense (complete silencing) or antisense (incomplete) strand of the RNaseP sequence, were plated onto EMJH media containing spectinomycin. Colonies obtained from two independent experiments were counted. (B)Validation of essentiality of ncRNA RNaseP was demonstrated by inducible silencing. Cells harboring the pMaOri.Inducible:dCas9 alone or with the sgRNasePsense were seeded onto EMJH plates with spectinomycin without (-) or with ( +) IPTG. Two independent experiments were performed and at each, two clones of conjugative E. coli were used. Number of colonies recovered in the absence or presence of IPTG was compared by t-test (*p < 0.05).