Researcher(s)
- Muhammad Burki, Biological Sciences, University of Delaware
Faculty Mentor(s)
- Vijay Parashar, Medical and Molecular Sciences, University of Delaware
Abstract
Bacteria protect themselves from bacteriophage infection using diverse innate-immune systems. One rapidly expanding family is CBASS (cyclic oligonucleotide-based antiphage signaling system), in which a nucleotidyl-cyclase sensor detects infection and produces a cyclic dinucleotide second messenger that activates a downstream effector to kill the infected cell before phage progeny can spread. A newly identified CBASS variant, CBASS-DAC, is unusual because its sensor is predicted to be a DAC (DisA-like adenylate cyclase) enzyme — a domain family previously associated with sporulation and DNA-damage sensing in Firmicutes, not antiphage immunity. The signal it generates, and how its uncharacterized N-terminal sensor domains (DACNG and DACNH) regulate cyclase activity, remain unknown.
To enable biochemical and structural characterization of CBASS-DAC, we designed a panel of four expression constructs spanning the full-length enzyme and three truncations that dissect the sensor and catalytic regions: full-length (1–581), DAC catalytic core (330–581), DACNH+DAC (234–581), and the sensor module alone (1–336). Each construct was designed with 18–21 bp homology arms for NEBuilder HiFi assembly into a BamHI/NotI-cut expression vector, with an N-terminal cleavable affinity tag. The full-length coding sequence was synthesized by Twist Biosciences and used as a template for PCR amplification of each construct with Phusion and Zymo Taq polymerases, followed by column purification.
To date, the full-length insert has been successfully amplified, purified, and prepared for ligation. HiFi assembly and transformation into chemically competent E. coli is underway, followed by colony PCR and diagnostic BamHI/NotI digestion to confirm correct insert integration. Verified clones will be sequence-validated and moved to protein expression trials, enabling downstream purification, activity assays, and cryo-EM studies to define the mechanism of this novel antiphage signaling enzyme.