show Abstracthide AbstractThe aim goal of our research was to generate a novel organism with a defined genome. We enlarged different bacterial cells for microinjection of various substances, for example, heterogeneous or designed genomic DNA. In order to generate a novel organism with an exogenous genome, it is essential to replicate exogenous genome DNA. In this study, we elucidate the timing of replication arrest in the process of bacterial protoplasts or spheroplasts enlargement. Among the enlarged bacterial cells in our laboratory, Enterococcus faecalis and Lelliottia amnigena enlarged to a micro-injectable size. Therefore, we used these bacterial spheroplasts or protoplasts.