Extended Data Fig. 7: Analysis of the effects of SPARDA on cell viability and nucleic acid degradation in vivo.
a, SYTO 9 and propidium iodide (PI) staining of E. coli cultures expressing SPARDA or containing control empty pBAD. Cell cultures were grown for 5 and 7 hours after induction in experiments with NbaSPARDA (left column) and CmeSPARDA (right column), respectively. Strains containing the second interfering plasmid pCDF were grown under identical conditions. The cells were collected by centrifugation, stained with SYTO 9 and PI, and analyzed by microscopy. The scale bar is 10 μm. The experiment was independently repeated 3 times, producing similar results. Representative fields of view (from 5–7 analyzed in each experiment) are shown. b, Analysis of the integrity of plasmid DNA in E. coli strains expressing CmeSPARDA (lanes 6–7, 11–12) or containing a control empty pBAD plasmid (‘E’, lanes 3–4, 9–10), in the absence (lanes 3–4, 6–7) and in the presence (lanes 9–12) of pCDF. Plasmid DNA was purified at 7 h of growth after induction of CmeSPARDA. For each condition, the results of two replicate experiments are shown. Lanes 2, 5 and 8 contain control samples of intact pBAD, pBAD-CmeSPARDA and pCDF. c, Purification of plasmid DNA from E. coli strains containing empty pBAD (‘E’, lanes 3–4), or pBAD encoding NbaSPARDA (WT or CD, lanes 6–9), or pBAD together with pCDF or pACYC (lanes 11–14 and 16–17, respectively) at 6 h of growth after induction of SPARDA expression (0.1% Ara). The results of two replicate experiments are shown. Lane 1 contains length marker, lanes 2, 5, 10 and 15 contain control samples of intact pBAD, pBAD-WT NbaSPARDA, pCDF and pACYC184. d, Analysis of total RNA purified from E. coli cultures containing pBAD (empty ‘E’, or encoding WT or CD NbaSPARDA), or pBAD together with pCDF, at 7 h of growth after induction of NbaSPARDA. Positions of 23 S and 16 S rRNA are indicated. A representative gel from two independent experiments, which produced similar results. Lane 1 in panels b-d contains length marker.
