Making bacterial evolution visible in real time: chemistry comes to the aid of biology

The protocol incorporates an in silico validation phase (Phase 1) with explicit GO/NO-GO/PIVOT criteria, which permits the filtering of non-viable substrates before the expenditure of substantial experimental resources — an exemplary practice in experimental design.

The hypothesis proposes an elegant and original integration of ABPP (Activity-Based Protein Profiling) with directed evolution, creating a direct and quantifiable link between individual enzymatic activity (fluorescence) and fitness at the population level. This surpasses indirect growth-based measures of fitness.

The idea of directly coupling enzymatic activity to a fluorescent signal via bioorthogonal chemistry is conceptually elegant and could, in theory, permit real-time monitoring of evolution.

A niche but well-identified market: R&D groups in directed evolution at actors such as Codexis, Ginkgo Bioworks, or Arzeda, which today pay millions for blind selection cycles, would have an immediate interest in a system that renders the evolution of an enzyme visible in real time. The time saved on variant optimisation (e.g., for industrial enzymes or biocatalysts) is a direct selling point.

A mechanistically elegant hypothesis is proposed, featuring an innovative coupling between evolutionary selection and real-time phenotypic readout via bioorthogonal chemistry (ABP + click chemistry).