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...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.展开更多
基金our funding sources:US Office of the Secretary of Defense Applied Research for the Advancement of S&T Priorities program Synthetic Biology for Military Environments and the US Army Combat Capabilities Development Command Chemical Biological Center Section 2363 Biological Engineering for Applied Materials Solutions program.This work was performed while Marilyn Lee held an NRC Research Associateship award at US Army CCDC CBC.
文摘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.
