Introduction New treatments for colorectal cancer (CRC), the world’s second deadliest cancer, are possible with tumour-homing bacteria. Bacterial genetic engineering can produce safe, programmable ‘micro-factories’ for tumour-specific drug delivery. However, as we reviewed, bacteria rapidly evolve1, and tumour-specific adaption is uncharacterised.  We previously described tumour-homing capabilities of Escherichia coli Nissle 1917 (EcN)2. This project aims to characterise tumour-specific EcN evolution in CRC and develop toxin-producing strains with synchronised lysis circuits (SLC) for targeted tumour treatment. SLCs allow synchronised lysis at high population density. EcN regrowth from chance survivors causes cyclical toxin release.   Methodology EcN was orally administered to CRC-bearing mice and reisolated from colonised tumours after two weeks, then serially passaged through four more CRC-bearing cohorts. Tumour-evolved and parental isolates underwent whole genome sequencing (WGS) to identify adaptive mutations. Toxin-encoding and SLC plasmids were introduced to EcN. Cytotoxicity of media conditioned with lysing or non-lysing toxin-EcN strains was tested on CRC organoids.    Results WGS of tumour isolates identified recurrent mutations in key EcN genes. Modelling variants and phenotypic analyses suggested mechanisms of action promoting tumour colonisation. Western Blotting confirmed toxin expression and SLC-dependent release. Conditioned media from toxin-EcN cultures showed remarkable SLC-dependent toxicity against patient-derived CRC organoids.    Conclusions Toxin-EcN strains are highly cytotoxic, with lysis-dependent payload release, which supports tumour-specific treatment. We also identified tumour-specific adaptations that could be incorporated into toxin-EcN chassis to improve tumour-homing for future translational studies.   References 1. Leeflang, J. et al. npj Biofilms and Microbiomes, 2025. 2. Gurbatri, C. et al. Nature Communications, 2024.