Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1(BBa_K4960021):

This is a basic part to enables the delivery of UCP1 by serving as the payload for the PVC delivery system. UCP1 is a naturally occurring mitochondrial uncoupler protein found in brown adipose tissue of mammals. Previously, we observed an unexpected interaction between the Pdp1NTD domain and UCP1 , which could possibly change the local protein structure and affect the translocation and function of UCP1. With the same protocol, we predicted the structure of a few design options and found that we could simply solve this problem by swapping UCP1 and EGFP .

Core expression cassette of pPayload plasmid to generate PVCs carrying Pdp1NTD-EGFP-UCP1 (BBa_K4960031):

This part is a composite part containing five coding genes that are responsible for payload packaging and the regulating the PVC assembly (Figure 2). Herein, the fusion protein Pdp1NTD-3*GGSGG-EGFP-2*GGGSG-UCP1 (encoded by BBa_K4960021) is loaded into the PVC system as a payload protein; PAU RS24015 (BBa_K4960017), PAU RS16560 (BBa_K4960018), PAU RS16565 (BBa_K4960019), and PAU RS16570 (BBa_K4960020) are regulatory genes play an integral role in the assembly of PVC systems. Similarly to the work published by Kreitz et al[1]. By electroporating E. coli cells with both a pPayload plasmid carrying Pdp1NTD-EGFP-UCP1 payload and a pPVC plasmid carrying E01DARPin (pNC090), we got a particle that specifically targets EGFR-expressing cells and delivers Pdp1NTD-EGFP-UCP1 protein. It can boost cellular energy expenditure by specifically deliver UCP1 into target cells.