100 generation
The -PULSE-0 strainwas cultivated in 20 mL of CSM medium using the Chi.Bio turbidostat platform in dither mode, starting at an initial OD600 of 0.1. In this setup, cells were repeatedly grown to an OD600 of ~0.6, at which point they were diluted back to an OD600 of 0.3. Each of these growth-dilution cycles was defined as one generation. The reactor was operated until more than 100 dilution steps had occurred. Genomic DNA was extracted from both the -PULSE-0 preculture and the culture after the passage of 100 generations. The four PULSE promoters were amplified using the KAPA HiFi HotStart ReadyMix PCR kit (Roche, Basel, Schweiz) with the appropriate primer pairs. The PCR products were purified using Agencourt AMPure XP beads (Beckmann Coulter, Brea, USA) and analyzed on a 2% agarose gel. Further, the amplicons were prepared for Nanopore sequencing according to the Ligation Sequencing DNA V14 (SQK-LSK114) protocol provided by Oxford Nanopore Technologies (Oxford, UK). The library was loaded onto two Flongle flowcells (FLO-FLG114) (Oxford Nanopore Technologies, Oxford, UK). The sequencing runs lasted 24 h and generated 360 k reads, with N50 of 958 b. Base calling of the Nanopore sequencing run was performed by the High-accuracy model v4.3.0, 400 bp"