History-Dependent Adaptation to Lethal Genetic Modification under Antibiotic Exposure Yuta Koganezawa (1), Miki Umetani (1, 2), Moritoshi Sato (1, 2, 3), Yuichi Wakamoto (1, 2, 3) 1. Graduate School of Arts and Sciences, the University of Tokyo, 2. Research Center for Complex Systems Biology, the University of Tokyo, 3. Universal Biology Institute, the University of Tokyo Genetic modification such as gene deletion and mutations could cause significant changes in cellular phenotypes and even cell deaths. However, it remains unexplored how rapidly individual cells transit to a new phenotype in response to genetic modification and whether cellular phenotypes always converge to identical states in given environments irrespectively of the historical details such as when the modification was introduced. To address these questions, we conducted single-cell analysis combining the Mother Machine microfluidic device (P. Wang et al. Curr. Biol., 2010) and the Magnet-Cre light-inducible gene-recombination system (F. Kawano et al. Nat. Chem. Biol., 2016). We provoked pre-designed deletion of fluorescently-tagged and chromosomally-encoded chloramphenicol resistant gene, chloramphenicol acetyltransferase (cat), in Escherichia coli at arbitrary timings by blue-light activation of Magnet-Cre and observed the multi-generational dynamics of hundreds of individual cell lineages. The results showed that 20-30% of E. coli cells exposed to 30-min blue light illumination lost the cat gene. Surprisingly, 40% of the cells whose cat gene was deleted under the constant exposure of a lethal dose of chloramphenicol (Cp) recovered and continued stable growth and division even without the resistance gene after initial responsive growth suppression. On the other hand, the cells whose cat gene was deleted 10 hours or longer before Cp exposures failed to exhibit such growth recovery and continuation. Therefore, the cells with a non-resistant genotype can survive and adapt only when the gene deletion occurred under or immediately before the drug exposures. To investigate intracellular state transitions after cat gene deletion, we observed a strain expressing two fluorescently-labeled ribosomal proteins, RplS-mCherry and RpsB-YFP (R. Nikolay et al., Nucleic Acids Res., 2014), from the native chromosome loci. We found that an expression balance of the ribosomal proteins collapsed in all the cells in response to cat-gene deletion but recovered in the adapted cell lineages along with growth restoration. The recovery occurred gradually in the timescale of over 10 generations. These results demonstrate that a non-resistant genotype has the potential to generate resistant phenotypes through multi-generational state transition. However, its access is limited by the historical conditions when the deletion was introduced.