UC San Diego

We developed the Multilayered Instrument for Continuous Adaptive Laboratory Evolution (MICALE), a novel apparatus for Adaptive Laboratory Evolution (ALE) of microbial strains with increased stress resistance. MICALE has two distinct media types separated by density. The less-dense top layer media (TLM) is permissive for growth of the wild type. A constant flow of media and removal of waste maintains a continuous culture in the upper region only. The denser bottom layer media (BLM) has an added stressor and remains static, allowing for batch-culture growth of an evolved strain. As they arise, mutants with stress-resistant phenotypes can colonize the lower regions. We tested MICALE’s ability to produce evolved strains by subjecting Escherichia coli MG1655 to high concentrations of sodium chloride (NaCl,) magnesium chloride (MgCl2,) or the antibiotic ciprofloxacin in the BLM. Measurements during experiments show that MICALE functions as intended; density stratification is maintained, and the media flow controls the population size in the upper layer. We tested for stress-tolerant phenotypes by measuring the optical density (OD600) of MICALE samples alongside the ancestor in increasing amounts of stressor. From the resulting OD600 curves, we extracted values representing stress-resistance using a novel analysis developed for atypical growth curves. We then performed whole genome resequencing followed by breseq to identify mutations linked to observed phenotypes. Contamination, however, prevents us from determining whether MICALE is effective at producing strains with increased stress tolerance. Our work provides a foundational methodology and outlines improvements for future ALE experiments to successfully evolve stress tolerant strains using MICALE.