Expression of an entire genome in an acellular heterologous system – ExEGeSyS

Synthetic Biology is an emerging scientific field, combining advanced knowledge of biological systems with concepts borrowed from chemistry, engineering and computer science. Its global objective is to design and build synthetic life-like or living systems. One of the strategies to reach these ambitious goals is called “Bottom-Up”. It proposes to use a constructive approach in which cells will be reconstituted by the controlled assembly of an array of synthetic components.
Important conceptual steps have already been made toward the Bottom-Up production of these “cell-like compartments”, predominantly through the co-encapsulation of plasmids and cell-free expression systems. These achievements in bio-system reconstitution indicate that the next logical step, the encapsulation of a complete genome, is now conceivable although highly challenging
Interestingly, this process of introducing a chromosome in a heterologous expression compartment to create a living organism has already been successfully achieved. Indeed, in Whole Genome Transplantation (WGT), a full bacterial chromosome can be transferred into a living, compatible cell, yielding a population of viable cells governed exclusively by the exogenous chromosome.
Successful WGT in mycoplasma proves that one can:
i) Transfer a chromosome in a compartment containing a heterologous expression system;
ii) Initiate the correct expression of the chromosome content (“Boot-up”);
iii) Create a cell governed by an exogenous genome.
Given the parallels between Bottom-Up and WGT, we propose to use WGT as a model to understand the general rules governing the interactions between a genome and a recipient compartment in a cell-free system. In particular, we want to explore the determinants of the compatibility between the Donor genome and the Recipient machinery. We hypothesize that a key parameter is the ability of the Recipient machinery to “interpret” (transcribe/translate) the data encoded on the Donor chromosome. Specifically, we identify as a potential critical step of the Boot-up, the synthesis and correct assembly of the Donor expression machinery (RNA polymerase, transcription factors, ribosomes, etc…), which would then take over the whole expression of the Donor genome.
The main objective of the ExEGeSyS project is thus to use WGT to study the Expression of an Entire Genome in an acellular heterologous System. To reach that goal, we propose the following methodology:
We will use the components of the successful in vivo WGT to set up an in vitro experimental system called Whole Genome Expression Assay (WGEA). In WGEA, a complete chromosome corresponding to the genome used as Donor in WGT will be incubated in a cellular extract prepared from the cells used as Recipient in WGT. The genetic expression in the extract will be monitored in both a gene-specific manner using RT-qPCR and Western-Blot; and in a target-agnostic approach using RNA-sequencing and proteome characterization by Mass Spectrometry
We will also perform WGEA experiments in which both the Donor and the Recipient genomes are added to the Recipient cellular extract. Comparison of the expression data gathered for these “dual genome” experiments with the “Donor only” experiments described above will allow to better understanding the role of the Recipient genome.
Based on the WGT success/failure modes identified through the previous experiments, we will test the impact of “enhancers” on the Boot-Up. These molecules, added directly to the cellular extract or encoded by the Donor genome, should improve the recognition of the genetic information by the expression machinery.
Finally, we will work towards the miniaturization of our experiments, transitioning from bulk reactions (microliter scale) to compartmentalized reactions (nanoliter scale) through the packaging of Donor genome in micron-sized synthetic compartments.