Journal: Nature Communications

Article Title: Synthetically designed anti-defense proteins overcome barriers to bacterial transformation and phage infection

doi: 10.1038/s41467-026-74301-8

Figure Lengend Snippet: a Top, the DdmDE system of V. cholerae O1 El Tor used in this study. Bottom, surface representation of the DdmDE heterodimer (PDB ID 9EZY) . Domain locations are indicated. Guide DNA (red) and plasmid DNA (yellow) are shown. b Schematic representation of the experimental set up. The starting point is a library of E. coli bacteria with or without DdmDE, carrying the pool of plasmids, each expressing a single candidate inhibitor. Cultures of these bacteria were grown for 50 generations and plated on antibiotic-containing media. Colonies that grew were collected, and their plasmids were sequenced. c Enrichment was calculated by dividing reads per candidate gene in the initial library by the reads per candidate gene at the end point of the experiment. Read counts were normalized to total reads per sample. Top, no enrichment of candidate plasmids in control cells. Bottom, enrichment of candidate plasmids in DdmDE-expressing cells. Shown is a representative of three replicates. All replicates are shown in Fig. . d Plasmid retention in E. coli cells heterologously expressing the V. cholerae DdmDE system. Plasmid retention is calculated by dividing CFUs (colony forming units) obtained on antibiotic-containing media by CFUs on non-selective media after 50 generations of growth. Control is an RFP-expressing plasmid. Bar graphs represent the average of three biological repeats, with individual data points overlaid. e Surface representation of the DdmDE complex modeled with the verified inhibitors. Structures for DdmDE binders 1-4 were modelled via AF3. DdmDE binder 5 was modeled with a cropped section of DdmE and then aligned to the complex structure (Methods) ( f ) As in panel d , plasmid retention assays were performed in V. cholerae O1 El Tor, which naturally encodes the DdmDE system, using either an RFP-expressing control plasmid or plasmids expressing each of the top four binder candidates. Bar graphs represent the average of three biological repeats, with individual data points overlaid.

Article Snippet: Candidate DdmDE binders were ordered from Twist Bioscience as a ssDNA oligo pool with primer binder sites for PCR amplification and downstream Gibson assembly.

Techniques: Plasmid Preparation, Bacteria, Expressing, Control