Methodologies for preparation of prokaryotic extracts for cell-free expression systems

Cell-free systems that mimic essential cell functions,such as gene expression,have dramatically expanded in recent years,both in terms of applications and widespread adoption.Here we provide a review of cell-extract methods,with a specific focus on prokaryotic systems.Firstly,we describe the diversity of Escherichia coli genetic strains available and their corresponding utility.We then trace the history of cell-extract methodology over the past 20 years,showing key improvements that lower the entry level for new researchers.Next,we survey the rise of new prokaryotic cell-free systems,with associated methods,and the opportunities provided.Finally,we use this historical perspective to comment on the role of methodology improvements and highlight where further improvements may be possible.

Silk fibroin as an additive for cell-free protein synthesis

Cell-free systems contain many proteins and metabolites required for complex functions such as transcription and translation or multi-step metabolic conversions.Research into expanding the delivery of these systems by drying or by embedding into other materials is enabling new applications in sensing,point-of-need manufacturing,and responsive materials.Meanwhile,silk fibroin from the silk worm,Bombyx mori,has received attention as a protective additive for dried enzyme formulations and as a material to build biocompatible hydrogels for controlled localization or delivery of biomolecular cargoes.In this work,we explore the effects of silk fibroin as an additive in cell-free protein synthesis(CFPS)reactions.Impacts of silk fibroin on CFPS activity and stability after drying,as well as the potential for incorporation of CFPS into hydrogels of crosslinked silk fibroin are assessed.We find that simple addition of silk fibroin increased productivity of the CFPS reactions by up to 42%,which we attribute to macromolecular crowding effects.However,we did not find evidence that silk fibroin provides a protective effects after drying as previously described for purified enzymes.Further,the enzymatic crosslinking transformations of silk fibroin typically used to form hydrogels are inhibited in the presence of the CFPS reaction mixture.Crosslinking attempts did not impact CFPS activity,but did yield localized protein aggregates rather than a hydrogel.We discuss the mechanisms at play in these results and how the silk fibroin-CFPS system might be improved for the design of cell-free devices.