The RNADarkMatter project aims to develop an in vitro platform to express and study gene expression in model and non-model bacteria, both cultivable and non-cultivable, from the human gut microbiome.
Non-cultivable organisms represent the vast majority of gut bacteria and are poorly understood. The diversity of growth conditions, behaviors, and cell compositions makes standard gene expression and protein production protocols ineffective with these non-model bacteria limiting our understanding of these organisms.
I propose to use cell-free systems and build a collection of plasmids designed to emulate the transcription machinery of human gut microbiota organisms. The cell-free systems consist of a mixture containing a cell lysate supplemented with an energy buffer and amino acids. As the transcription and translation processes remain active, proteins can be produced directly by adding DNA to the above mixture. The protocol requires large volumes of cell culture that cannot be obtained with difficult to culture or non-culturable organisms in the laboratory. However, the cell-free system can produce transcription machinery from non-model organisms, which will be able to express transcription units from organisms of interest.
The objective of this platform is to express complete or partial genomes (natural, PCR amplified or synthesized) in a systematic and controlled manner. I will measure transcriptomes expressed under a wide range of conditions (different sigma factors, transcriptional regulators). This project will lay the foundation for functional genomics on organisms for which only the genetic sequence is known.
