Building a cell from the ground up would help identify the minimal set of elements necessary for a cell-like compartment to become a functional living entity. This requires an extensive understanding of the contribution of each component to the cell function. This project focuses on genetic information and its processing by a compartment capable of gene expression, using an original approach named whole genome transplantation (WGT). This technique consists in isolating a whole bacterial genome belonging to Species A (donor genome) and installing it in the cytoplasm of Species B (recipient cell), resulting in cells genotypically and phenotypically identical to Species A. WGT is currently performed on Mollicutes, the simplest living forms capable of autonomous replication outside of a host, an ideal model for studying essential requirements for life. Understanding what defines the compatibility between the donor genome and recipient cell may lead to identifying key elements that enable booting up a living cell and to understanding the rules that regulate the interactions between genetic material and the compartment which expresses it. We hypothetize that the ability of the recipient transcription machinery to “interpret” the data encoded on the donor chromosome is essential for successful boot-up. Our approach consists in engineering a recipient cell, Mycoplasma capricolum (Mcap), to preload it with transcription factors belonging to a donor genome, Mesoplasma florum (Mflorum). The coding sequences of the five subunits of the Mflorum RNA polymerase were cloned into plasmids along with their native promoters. Transformation of Mcap suggests that the Mflorum genes can be expressed individually in Mcap and that their presence does not impact the recipient’s survival. In the next phase, the 5 subunits will be simultaneously expressed in Mcap. The resulting cells will be used as recipients for WGT assays and structural analysis of their transcription machinery will be performed.Read less <