Recently, new approaches referred to as Synthetic Biology (SB) have emerged. The work done at the J. C. Venter Institute (JCVI) has significantly contributed to the development of SB by showing: (1) the synthesis of complete bacterial genomes starting from chemically produced oligonucleotides, (2) the genome-scale engineering using yeast as a platform, (3) the transplantation of isolated bacterial genomes to other bacteria or from yeast to bacteria. Mycoplasma capricolum subsp. capricolum (Mcap) and Mycoplasma mycoides subsp. capri (Mmc), which are related species, were used as recipient cell and donor genome, respectively. Mycoplasmas belong to the class Mollicutes, a group of wall-less bacteria phylogenetically related to low G+C% Firmicutes. Mycoplasmas are characterized by the use of UGA as a tryptophan codon and a small genome. Mollicutes include several pathogens affecting humans, animals and plants. One of the limiting factors to better understand the molecular basis of their pathogenicity is the lack of efficient genetic tools. The goal of SYNBIOMOLL is to extend Synthetic Biology key technologies to other Mollicutes species of interest. Because several bacterial genomes have already been cloned in yeast and the synthetic assembly of large DNA fragments is in current use, the major technological limitation to a much wider use of these SB tools is our ability to extend the genome transplantation technology to other microbial species. In task 2 (task 1 is the coordination of the project), we propose to study the mechanisms of genome transplantation by evaluating the degree of relatedness necessary between a donor cell and a recipient cell and also by identifying some of the genetic factors responsible for this compatibility. First, we will transplant in Mcap recipient cell the genomes from species with increasing phylogenetic distance from the recipient cell to identify the transplantation compatibility limit. Then, using a troubleshooting approach, we will determine whether non-compatibility is a problem of replication and/or expression of the donor genome in the recipient cell. Specific genome regions, including the chromosomal replication origin, will be exchanged between Mcap and a non-compatible genome to evaluate the impact on transplantation. As a control, the same exchanges will be performed with a compatible genome. For this sub-task, genomes will be cloned into yeast to be modified accordingly. The expected results should help identifying trans-acting proteins and/or cis-acting sequences required to boot-up heterologous genomes into recipient cells. In task 3, we plan to develop genome transplantation in another phylogenetic group using Acholeplasma laidlawii (AL) as a recipient cell. AL can grow in much simpler culture media than mycoplasmas and is considered as an intermediate species between mycoplasmas and low G+C% firmicutes. Genome transplantations will first be performed with homologous donor genome and then with heterologous donor genomes from two other acholeplasmas. Once the conditions of genome transplantation in AL optimized, specific developments will be pursued to evaluate its capacities to replicate and express the genome of more distant non-cultivable, insect-transmitted plant-pathogenic phytoplasmas. The impossibility to cultivate these bacteria is a major barrier to their study and the development of plant protection strategies. In the SYNBIOMOLL project, we will evaluate AL ability to recognize and initiate replication of phytoplasma chromosomes using phytoplasma oriC plasmids and by replacing the oriC of AL with that of the phytoplasma. We will also use proteomic analyses to evaluate the expression of phytoplasma genes in AL. Succeeding genome transplantation in AL would be important per se but also would serve as a launching pad for the culture and the study of non-cultivable phytoplasmas. At the end of SYNBIOMOLL, Carole Lartigue intends to propose this project for an ERC Consolidator Grants.