Extended Data Fig. 2: Biochemical analysis of in vitro-loaded PVCs.
From: Targeted delivery of diverse biomolecules with engineered bacterial nanosyringes
a, Pvc10-cargo fusion proteins self-associate with Δpvc10 PVCs in vitro. Pvc10 alone or with a cargo (GFP) were incubated with PVCs lacking Pvc10, purified using ultracentrifugation, and visualized with an immunoblot. Only intact Pvc10 or Pvc10-GFP successfully purified with the PVC complex after the complementation reaction; –Pvc10 variants of both (harboring a scrambled Pvc10 sequence) failed to purify. All proteins were tagged with FLAG; bands were visualized with anti-FLAG immunoblot. Baseplate (Pvc12) served as a loading control. b, ssODNs can be loaded onto the PVC spike tip in vitro. ssODNs (labeled with Cy3) were titrated against PVCs containing Pvc10-HUHe (tagged with FLAG), and protein-DNA conjugation was assessed using a dual Cy3/FLAG blot. Only ssODNs containing the 13-nt HUHe recognition sequence produced an increase in molecular weight in both the DNA and protein bands, indicating these species became covalently linked.
