Paper 28 Details

Title

Ribosome synthesis and construction of a minimal cell using a cell-free expression platform

Full Paper Download

Download via this paper's page on the MIT Press ECAL 2015 Proceedings website.

Abstract

The creation of wet artificial life in the laboratory is a nontrivial challenge for biologists, chemists, and computer scientists. Such a challenge revolves around the modular integration of complex reactions networks to obtain functional biochemical units able of self-replication, self-reproduction, spatial-temporal control and ultimately open-ended evolution, e.g. minimal and artificial cells. As a step towards building minimal cells, we have developed a cell-free expression system for bacterial ribosome synthesis named iSAT: integrated Synthesis, Assembly and Translation for in vitro construction of Escherichia coli ribosomes. The ribosomal RNA, transcribed from its natural operon, selfassembles with ribosomal proteins added to the reaction mixture. Afterwards, in vitro built synthetic ribosomes translate a reporter gene. Such system is important to design ribosome with new functions, and for the bottom-up construction of a minimal cell.

Ribosome cell-free synthesis is an essential process for building a minimal cell that can maintain itself. Indeed, regeneration of encoded DNA molecular machineries, through a compartmentalized reaction network, will be necessary to ensure gene expression after cycles of self-reproduction. In this work, we have sought to improve the efficiency of the iSAT reaction to achieve the break-even milestone of ribosomes that are capable of constructing ribosomes (7,434 peptide bonds are needed to make a complete set of rproteins). To do this, we prepared and optimized the iSAT reaction system using a robot for liquid handling. The open nature of cell-free expression platforms enables precise settings of each component level for an optimal system’s configuration. Previously, high-throughput screening and machine learning have been used for the optimization of a cell-free protein synthesis and a liposomal drug formulation.

Here, I will present the optimization of in vitro ribosome construction using a cell-free expression system, and I will introduce future directions of the project. In particular, I will describe biochemical experimental spaces underlying the cellfree ribosome synthesis. I will show results on the optimization using a liquid handling robot for high-throughput experimentation. Our cell-free protein synthesis platform is the only one enabling in vitro ribosome construction, which is relevant to the synthesis of a minimal cell. Adding effective energy regeneration modules is also important for minimal cell projects. Therefore, I will also present data highlighting the ability to regenerate ATP with a non-phosphorylated energy substrate with the iSAT system. Recently, I developed a novel metabolic scheme for a minimal cell. The system is based on the catabolism of polysaccharides and/or a polyphosphate to regenerate ATP. Proteins are synthesized using a custom-made amino acids mixture.

The system is improved by overcoming a fundamental limitation: the efficient recycling of the orthophosphate (iP), which is the by-product of protein synthesis. As a result, ATP (adenosine triphosphate) is kept at steady state and available for in vitro transcription and translation. Currently, it represents the most powerful in vitro protein synthesis systems. One important feature of a minimal cell is the compartment or container, which more than physically interlock components and sub-systems, confers the necessary genotype-phenotype linkage for evolution. The compartment of the minimal cell is based on liposomes, and evolutionary dynamics such as fusion and division, are important for resource feeding and selection respectively.

In summary, the work described is designed to lay the foundation for the construction of a synthetic replicating entity by building up synthetic biological unit operations (e.g. cell-free synthesis of constituent parts) and fine-tune the starting blueprint. Indeed, through the bottom-up synthesis of a minimal cell, we are building and understanding complex biological systems.