Synthetic biology aims to create a viable synthetic cell. However, to achieve this goal, it is essential first to gain a profound understanding of the cellular systems used to build that cell, how to reconstitute those systems in the compartments, and how to track their function. Transcription and translation are two vital cellular systems responsible for the production of RNA and, consequently, proteins, without which the cell would not be able to maintain itself or fulfill its functions. This review discusses in detail how the Protein synthesis Using Recombinant Element (PURE) system and cell lysate are used to reconstitute transcription–translation in vitro. Furthermore, it examines how these systems can be encapsulated in GUVs using the existing methods. It also assesses approaches available to image transcription and translation with a diverse arsenal of fluorescence microscopy techniques and a broad collection of probes developed in recent decades. Finally, it highlights solutions for the challenge ahead, namely the decoupling of the two systems in PURE, and discusses the prospects of synthetic biology in the modern world.

Single-stranded nucleic acid (ssNA) binding proteins must both stably protect ssNA transiently exposed during replication and other NA transactions, and also rapidly reorganize and dissociate to allow further NA processing. How these seemingly opposing functions can coexist has been recently elucidated by optical tweezers (OT) experiments that isolate and manipulate single long ssNA molecules to measure conformation in real time. The effective length of an ssNA substrate held at fixed tension is altered upon protein binding, enabling quantification of both the structure and kinetics of protein–NA interactions. When proteins exhibit multiple binding states, however, OT measurements may produce difficult to analyze signals including non-monotonic response to free protein concentration and convolution of multiple fundamental rates. In this review we compare single-molecule experiments with three proteins of vastly different structure and origin that exhibit similar ssNA interactions. These results are consistent with a general model in which protein oligomers containing multiple binding interfaces switch conformations to adjust protein:NA stoichiometry. These characteristics allow a finite number of proteins to protect long ssNA regions by maximizing protein–ssNA contacts while also providing a pathway with reduced energetic barriers to reorganization and eventual protein displacement when these ssNA regions are diminished.