Cell Free Synthesis of Protein Microcompartments

David C. Garcia & Mitchel J. Doktycz

Cell free synthesis (CFS) has developed into a technique with diverse uses. One emerging application utilizes CFS to design and build microscale structures with potential uses in biotechnology. For example, bacterial microcompartments (BMCs) are self-assembling bioreactors that house metabolic functions and regulate access of substrates and metabolites. The use of BMCs to contain non-native enzymes and substrates could develop into a technique for creating chemicals and pharmaceuticals. Cell free synthesis approaches can be valuable for engineering the targeting and assembly of enzymes into synthetic microcompartments. Here, we report on the production of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment components in an E. coli based cell free environment. Recombinant expression of the EutS microcompartment proteins are being generated to form polyhedral compartments. We have designed a system to co-express EutS and sfGFP tagged with an encapsulation peptide which can facilitate the targeting of sfGFP to the protein shell. These experiments will demonstrate a proof of concept for using bacterial microcompartments as nano-bioreactors for diverse applications.