Scientists believe that before complex cellular life emerged on Earth, simpler “containers” may have served as early "protocells." Droplets containing various chemical systems are one candidate for such cell-like containers, but we currently have a poor understanding of whether such a protocell could have grown and divided—a fundamental requirement for life. Using a technique called electrochemistry, which applies electric current to drive chemical reactions, Professor Julio Alvarez will study how electrical charges affect the behavior of tiny droplets. When droplets become sufficiently charged, they can become unstable and split apart, potentially mimicking cellular division. The research team will use ultra-small electrodes to precisely control chemical reactions that modify droplet charge states. They'll examine various factors including different surfactants (molecules that stabilize droplets), solution environments, and conditions that mimic the crowded, viscous environment inside modern cells. By understanding what controls droplet growth and division, this research could illuminate a critical step in the transition from non-living chemistry to life on early Earth. The findings may also contribute to modern synthetic biology efforts to build artificial cells from scratch.

This grant funds a collaboration between Sara Walker, a Professor in the School of Earth and Space Exploration and Deputy Director of the Beyond Center for Fundamental Concepts in Science at Arizona State University, and Leroy Cronin, the Regius Professor of Chemistry at the University of Glasgow, to advance the development of Assembly Theory (AT) by formalizing the mathematical structure of the theory and extending its applicability. Assembly Theory is a framework developed to quantify the complexity of molecules and objects by assessing the minimal number of steps required to assemble them from fundamental building blocks. The theory assigns an assembly index to objects, which serves as a measure of their structural complexity. It is one promising framework for quantifying, understanding, and predicting the emergence and evolution of varied types of complex objects. Walker and Cronin will work towards a general AT framework by determining a suite of mathematical relationships that hold across any assembly space. This will involves applying AT to other substrates, in this case, minerals and genomes / proteomes. The team will develop an assembly theory for minerals and they propose to trace out the evolutionary history of minerals on Earth by combining mineral AT with existing phylogenetic methods that reveal evolutionary connections between objects. The team will also apply AT to large molecules made from nucleic acid building blocks (e.g. DNA / RNA) and from amino acid building blocks (proteins). The plan is to combine AT with phylogenetic techniques to gain insights into the evolutionary history of the modern-day transcription and translation systems used by all known life. Walker and Cronin will also attempt to establish connections between assembly theory and thermodynamics. By developing a bridged framework—Assembly Thermodynamics—they expect to quantify how limits on free energy constrain when a selection phase transition takes place, and to make predictions about such transitions that can be tested in laboratory experiments. If successful, this project will uncover mathematical relationships that apply to all versions of AT (irrespective of the type of object undergoing complexification), allow AT to describe complex minerals and polymers (DNA, RNA, proteins), and make connections between AT and thermodynamics.

The information stored in genes plays a huge role in directing the cellular processes underlying life, but stored information alone is inadequate to explain how cells function. Cellular forces and associated motions also play a decisive role in determining whether and when genetic information is expressed because DNA can only be copied when a chromosome is in its decompacted state and because forces and other cellular dynamics drive the transition between compacted and decompacted chromosome states. Forces are also central to the creation and migration of chromosomal density fluctuations, pockets of compaction in a nominally decompacted chromosome region (or of decompaction in a compacted region). These migrating density fluctuations can, in different scenarios, contribute both to biological function and to disfunction. Funds from this grant support Andrew Spakowitz, a Professor of Chemical Engineering and of Materials Science & Engineering at Stanford University, to develop theoretical models that will advance our understanding of how forces/dynamics impact the organization and function of chromosomes in eukaryotic cells. Spakowitz plans to achieve a multi-scale model via a staged progression of model development that describes the coupled chemical/mechanical dynamics starting at the molecular scale, he will then expand to intermediate-scale chromosome dynamics (about a tenth of a chromosome), and end with an exploration of dynamics at the scale of an entire chromosome or group of chromosomes. Forces to be modeled include constraining forces that arise from chemical bond formation between two typically distant segments of a chromosome that happen to come into proximity owing to the wiggling motion of a chromosome in the aqueous environment of a cell and which, in turn, promote chromosome compaction; thermal agitation forces (owing to collisions with water molecules) that drive chromosome decompaction; and the force exerted on DNA (by RNA polymerase) during transcription (reading of genetic information).  if successful, the project will improve our understanding of how coupled mechanical and chemical interactions at the molecular-scale drive the organizational dynamics observed at the much larger length scale of a chromosome or group of chromosomes, thereby providing insight into how forces mediate access to and use of genetically encoded information.

Building a simple form of cellular life from scratch is an ambitious goal, and working towards that goal will advance our understanding of living systems. One promising strategy to achieve this goal is based on the intuition that by avoiding the highly evolved and complex biomolecules found in present day cells, it should be possible to build a (proto)cell that’s much simpler than a natural cell. Professor Katarzyna (Kate) Adamala, an Assistant Professor of Genetics, Cell Biology and Development at the University of Minnesota, takes a different view. She’s open to using any molecules at her disposal to build life from scratch, and her approach to circumventing the complexity of modern cells is -somewhat paradoxically- to dive into molecular complexity. Her thinking is that it should be possible to engineer a complex, multifunctional protein that does what natural cells use several proteins to achieve. If she’s right, then what’s achieved by a complex network of chemical reactions in a natural cell could be achieved by a much simpler set of reactions in a synthetic cell that leverages more complex proteins. This grant supports Professor Adamala’s work aimed at circumventing the complexity of the chemical networks found in present-day cells by engineering more complex proteins. Natural cells are limited to building a tiny fraction of all possible proteins; primarily because they’re restricted to using a small fraction (22) of the known amino acids (~500). Adamala will study what limits which amino acids natural cells use to build proteins, circumvent those limits, and engineer a protein synthesis system -and an associated synthetic cell- that can build a wider range of proteins than can be built by natural cells. Adamala and her team plan to learn about the limitations of natural protein synthesis and expand the chemical diversity of translation via three activities. First, they will evolve the standard ribosome so that it’s capable of building proteins from noncanonical amino acids (ncAAs), amino acids other than the 22 used by natural cells. Second, they will engineer a modified version of a certain protein that’s known to ‘rescue’ failed protein translation. The modified protein will be able to rescue stalled translation involving noncanonical amino acids. Third, the researchers will engineer an RNA translation system that incorporates up to 20 ncAAs. Adamala and her colleagues will use these three products to create a synthetic cell -a liposome vesicle encapsulating “cytoplasm”--that is capable of expanded translation.

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.

Unraveling how mechanical forces / physical effects contribute to biological function is an underexplored yet important aspect of understanding living systems. This grant provides continuing support to a trio of early-career researchers at Rockefeller University for a series of experiments geared at understanding how mechanical force impacts two important biological functions: the copying of information stored in DNA (transcription) and the coordinated development of a field of cells into a multicellular functional unit (here, skeletal tissue). The proposed transcription research will study how force impacts the dynamics and fidelity of the primary biomolecular machine responsible for transcription, RNA polymerase (RNAP). Forces will be applied to RNAP either using laser tweezers or via collisions between RNAP and various biomolecules that mimic RNAP collisions in live cells. Fluorescence microscopy will capture RNAP dynamics and cryogenic electron microscopy (cryo-EM) will reveal how the molecular-scale structure, and therefore the biochemical activity, of RNAP is modified by collisions. The tissue development research will use cryo-EM along with cellular biology methods that stimulate cell contraction -and thus force propagation- to study changes in molecular architecture that are driven by supracellular (beyond a single cell) force transmission.