← Experiments· 2026-06-25

E-01E-01 — Colibactin-null EcN chassis construction

planned

Remove the pks genotoxin island from E. coli Nissle 1917 to create a safe chassis for CNS deployment.

Hypothesis

E. coli Nissle 1917 (EcN) with dual deletion of pks genomic island genes clbP and clbS is a viable, fitness-neutral chassis — free of colibactin genotoxic activity and suitable for IND-enabling studies in a CNS context.

Background

EcN carries the pks genomic island, which encodes biosynthetic machinery for colibactin, a genotoxin that causes DNA double-strand breaks in host cells. In gut probiotic use, clinical significance is debated. For a therapeutic injected into neural tissue, any genotoxin is incompatible with regulatory submission — this deletion is non-negotiable before any downstream engineering proceeds.

ClbP activates colibactin for secretion. ClbS provides self-resistance. Dual deletion is the conservative approach for a CNS application. Kalantari et al. (2023, PLoS ONE, PMID 36730255) established that pks island deletion causes no growth defect in EcN.

This chassis is the substrate for all subsequent experiments. Nothing built on wild-type EcN is usable for regulatory purposes.

Design

  1. Construct deletion cassettes — CRISPR-assisted recombineering targeting clbP and clbS. Lambda Red recombineering is the validated method for precise deletions in EcN.
  2. Colony screening — PCR with primers flanking each deletion junction. Expected band shift confirms deletion at each locus.
  3. Sanger sequencing — Confirm junction sequences at both deletion sites.
  4. Whole-genome sequencing — Verify no off-target edits anywhere in the genome. Required for regulatory dossier.
  5. SOS reporter assay — Co-culture deletion strain alongside a DNA-damage SOS reporter (bacteria expressing a fluorescent reporter under SOS promoter control). Wild-type EcN will induce the response; deletion strain must not.
  6. Growth curve — Compare doubling time and final OD of deletion strain vs. wild-type EcN in LB and minimal media. Confirm no fitness cost.

Estimated cost: $8,000–$15,000 USD. Timeline: Weeks 1–6.

Readouts

  • PCR bands at correct sizes for clbP and clbS deletion junctions ✓ required
  • Sanger sequencing confirms expected junction sequences at both sites ✓ required
  • Whole-genome sequencing: no off-target edits detected ✓ required
  • SOS reporter: colibactin activity below detection limit in deletion strain ✓ required
  • Growth curve: no significant fitness cost vs. wild-type EcN ✓ required

Success criterion: all five readouts confirmed. This strain becomes the chassis for E-02 through E-07 and all downstream circuit integration.

Status notes

Planned. Awaiting CRO engagement to confirm BSL-2 capacity and microbial engineering capability. Smart Assays (Ness Ziona) is the primary CRO candidate under evaluation.