Models for Cell-Free Synthetic Biology: Make Prototyping Easier, Better, and Faster

Abstract

Cell-free TX-TL is an increasingly mature and useful platform for prototyping, testing, and engineering biological parts and systems. However, to fully accomplish the promises of synthetic biology, mathematical models are required to facilitate the design and predict the behavior of biological components in cell-free extracts. We review here the latest models accounting for transcription, translation, competition, and depletion of resources as well as genome scale models for lysate-based cell-free TX-TL systems, including their current limitations. These models will have to find ways to account for batch-to-batch variability before being quantitatively predictive in cell-free lysate-based platforms.

Multi-Enzyme Assembly on T4 Phage Scaffold

“…In our experiments, the three enzyme–Hoc constructs were incorporated into ΔHoc T4 in vitro . This type of in vitro synthetic biology has been exploited to circumvent drawbacks to cell-based approaches, such as toxicity and the presence of competing pathways (Koch et al, 2018 ; Sperl and Sieber, 2018 ). In addition, in vitro assembly allows better control over the stoichiometry of the enzymes in the multi-enzyme-scaffolded complex.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…Nevertheless, we still lack systematic studies on the influence of these different energy regeneration methods on lysate properties other than simple protein yield. In particular, for prototyping and characterization of circuits, it is known that resource competition leading to improperly balanced energy usage (Siegal-Gaskins et al, 2014;Koch et al, 2018), efficiency of energy sources and small molecule replenishment (Siegal-Gaskins et al, 2014;Borkowski et al, 2018), changes in binding kinetics due to magnesium ion concentration changes , and pH variability (Calhoun and Swartz, 2005b) are all dependent on the energy system used and are expected to have profound influence on circuit behavior.…”

Future Trends in Synthetic Biology—A Report

“…Combining this technology with DNA editing techniques, such as CRISPR/Cas9, will make the establishment of host production platforms and the generation of complex biosynthetic products easier and faster. One articulation of this could be in cell-free formats, in which the essential cellular machinery is reconstituted in vitro and used as a manufacturing platform (Villarreal and Tan, 2017; Koch et al, 2018).…”