Experimental tests of functional molecular regeneration via a standard framework for coordinating synthetic cell building

Abstract

The construction of synthetic cells from lifeless ensembles of molecules is expected to require integration of hundreds of genetically-encoded functions whose collective capacities enable self-reproduction in simple environments. To date the regenerative capacities of various life-essential functions tend to be evaluated on an ad hoc basis, with only a handful of functions tested at once and only successful results typically reported. Here, we develop a framework for systematically evaluating the capacity of a system to remake itself. Using the cell-free Protein synthesis Using Recombinant Elements (PURE) as a model system we apply our framework to evaluate the capacity of PURE, whose composition is completely known, to remake 36 life-essential functions. We find that only 23 of the components can be well tested and that only 19 of the 23 can be remade by the system itself; translation release factors remade by PURE are not fully functional. From both a qualitative and quantitative perspective PURE alone cannot remake PURE. We represent our findings via a standard visual form we call the Pureiodic Table that serves as a tool for tracking which life-essential functions can work together in remaking one another and what functions remain to be remade. We curate and represent all available data to create an expanded Pureiodic Table in support of collective coordination among ongoing but independent synthetic cell building efforts. The history of science and technology teaches us that how we organize ourselves will impact how we organize our cells, and vice versa.

Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System

“…One of the major challenges in cell-free synthetic biology or artificial cell synthesis is the construction of a reproducible artificial system. Recently, scientists have attempted to regenerate transcription and translation factors ,,− or other important cellular functions − through gene expression in cell-free systems to achieve reproducible artificial systems. However, the expression of these genes has not previously been coupled with DNA replication, which is an essential function of a reproducible system.…”

Reconfiguring the Challenge of Biological Complexity as a Resource for Biodesign

“…From its inception, biological complexity has been engineering biology’s central challenge ( 1 ). Biological systems involve seemingly uncountable, highly interconnected components, many of which remain poorly described and which are not necessarily fully decomposable ( 2 ). Engineering biology involves abstracting these complex systems into machinelike, discrete, interchangeable parts ( 3 ).…”

“…Biological complexity is a strength in these applications in that microbes—individually and communally—resiliently adapt to changing circumstances while maintaining a functional identity. As Wei and Endy have argued in describing where modularity fails in constructing living systems from nonliving parts, researchers (re)make systems in the image of what they expect them to be ( 2 ). Given the diversity of technical approaches now available, engineering biologists have choices: to erase complexity to make cells stupider, or to develop strategies to work with their intelligent complexity ( 27 – 29 ).…”

The Long Road to a Synthetic Self-Replicating Central Dogma

“…Since their conception by the Weissbach group in the late 1970s, partially purified CFE systems have presented viable solutions to confront the limitations of lysate-based systems. Ideally, a minimal CFE system is well-defined and composed solely of purified components and hence lends itself to being an easier model for understanding and manipulating life-like processes. , In particular, transcription–translation-coupled (IVTT) systems are the basis of efforts toward the construction of self-replicating, evolvable in vitro systems based on the central dogma. ,− In the past two decades, the reconstitution of PURE (protein synthesis using recombinant elements) systems (Figure ) from more than 35 proteins, ribosomes, tRNAs, and various small molecules has presented a viable basis for the development of cell-free systems capable of combined de novo synthesis of proteins, RNA, and even DNA. − However, PURE has considerable limitations when it is adopted to serve as the backbone of a self-regenerative MPC, − as it would need to be able to synthesize its genome, all of its own rRNAs, tRNAs, and protein components during its growth. ,,,, The minimum model for a self-replicating PURE system could be considered capable of self-replication once complete regeneration of its macromolecular components occurs by either (i) assuming an increasing volume (growth) or (ii) reaching a steady state between anabolism and catabolism that is far from thermodynamic equilibrium. Such a mode of self-replication should be readily compatible with strategies involving growth and division of artificial compartments that encapsulate a self-regenerative PURE system .…”

“…Such a mode of self-replication should be readily compatible with strategies involving growth and division of artificial compartments that encapsulate a self-regenerative PURE system . While current versions of PURE can synthesize fully functional proteins, ,,,, the expression levels of such proteins are not yet sufficient to sustain self-replication. Current estimates suggest that the expression capacity of PURE systems would have to increase at least 87-fold to achieve complete regeneration and ultimately self-replication of all PURE proteins .…”