An emerging global community of several hundred researchers has started to build cells from scratch by combining small molecules, purified proteins, lipids, and synthetic DNA to assemble simple synthetic cells from scratch. While this community has developed a wide range of ‘modules’ that implement various cellular processes and functions, the fact that these modules are developed independently (in different laboratories) is creating obstacles to progress. For instance, modules are intended to operate within a synthetic cell fluid (cytosol) but their creation across independent laboratories means that they are not developed in a standardized cytosol. This often leads to incompatibilities whereby the conditions that optimize the chemical performance of one module render another either non-functional or poorly performing. This grant to Richard Murray, a Professor of Control & Dynamical Systems and of Bioengineering at Caltech, and Akshay Maheshwari, co-founder and CEO of a company (b.next) “working to democratize synthetic cell engineering”, supports the continued development of Nucleus, a platform that provides a standardized suite of hardware components (chemicals / molecules) and procedures / recipes that can be used to build synthetic cells at varying levels of complexity. Murray and Maheshwari plan to develop three ‘modules’ that expand the technical capabilities of Nucleus, and to enclose the improved Nucleus cytosol within a lipid membrane to produce the first Nucleus synthetic cell. First, they will characterize the Nucleus cytosol performance (yield of transcription and translation) as a function of cytosol molecular composition, with the goal of identifying the molecular composition that best supports simultaneous operation of various modules. The Nucleus cytosol will be characterized across a wide range of various small molecule and protein concentrations, as well as under various conditions of RNA and ribosome abundance. They will also develop tools for implementing modules as ‘DNA constructs’, custom-designed DNA sequences that can be used to synthesize targeted proteins. Murray and Maheshwari will then develop three modules to improve the Nucleus cytosol. The first module aims to increases energy capacity by fostering energy recycling. The existing Nucleus cytosol uses certain energy-resource molecules that when metabolized produce toxins that accumulate and eventually poison the cytosol. The plan is to leverage a chemical pathway that uses a certain enzyme to regenerate the energy resource molecules from the toxins, thereby removing the toxins and replenishing energy resources.  The second module aims to control protein-expression through dynamic (time varying) control of protein abundance.  The existing Nucleus cytosol only allows for protein synthesis, with no ability to reduce protein abundance; a situation that amounts to a limitation on the ability to control the dynamics within a synthetic cell. The team will develop a module that uses a protein enzyme to continuously degrade targeted proteins and thereby provide temporal control over protein abundance. Lastly, the team will develop a ‘membrane-protein module’ and encapsulate the improved cytosol to create a Nucleus synthetic cell. The module will be used for inserting proteins into a synthetic cell lipid membrane; proteins that can transport nutrients into and waste out of a synthetic cell, as well as enable membrane-based cell-cell communication and implementation of various membrane-based molecular sensors. Finally, Murray and Maheshwari will encapsulate the improved membrane-module-enhanced cytosol and reoptimize the performance of the encapsulated cytosol to create a Nucleus synthetic cell. If successful, this project will lead to a novel, open-source platform for building synthetic cells that is accessed and further developed by the global synthetic cell community.