Endosymbiotic bacteria have evolved intricate delivery systems that enable these organisms to interface with host biology. The extracellular contractile injection systems (eCISs), are syringe-like macromolecular complexes that inject protein payloads into eukaryotic cells by driving a spike through the cellular membrane. In recent years, many synthetic biologists have paid more attention to a subtype of eCISs: the Photorhabdus virulence cassette (PVC). [1] At the same time, our team noted the increasing demand for weight-loss drugs in recent years, so we set our sights on Uncoupling protein UCP1, which has excellent potential and broad application prospects as a potential target for weight loss. In our registered and submitted parts, we provide a series of components using different combinations of parts to generate diverse PVCs and Payloads. Our favorite composite part Pdp1NTD-3*GGSGG-EGFP-2*GGGSG-UCP1->PAU_RS24015->PAU_RS16560->PAU_RS16565->PAU_RS16570(BBa_K4960031)is constructed with a Pdp1NTD-3*GGSGG-EGFP-2*GGGSG-UCP1(BBa K4960021) and four regulator genes: PAU_RS24015(BBa K4960017), PAU_RS16560(BBa K4960018), PAU_RS16565(BBa K4960019), PAU_RS16570(BBa K4960020).(Figure 1)

Referring to the work of Kreitz et al., PAU_RS16570-RS24015 (BBa K4960017-K4960020) has a regulatory effect on the assembly of PVC particles However, it is not clear what specific function each of the four genes has. We targeted one of them (PAU_RS16570) with site-specific mutation to silence its expression and purify PVC by packaging. The negative-stain transmission electron microscopy on purified PVCs showed without this gene, PVC cannot be assembled to function properly. (Figure 2) This result indicates that the four regulatory genes are essential for proper PVC folding. Therefore, we decided to retain the most complete structure for our project.

To validate the function of this part, We constructed pNC093, a pPayload plasmid carrying Pdp1NTD-EGFP-UCP1 payload . By electroporating E. coli cells with both pNC093 and a pPVC plasmid carrying E01DARPin (pNC090), we could then get a PVC_(EGFP-UCP1)^(EGFR-targeting) particle that specifically targets EGFR-expressing cells and delivers Pdp1NTD-EGFP-UCP1 protein (Figure 3a). To validate whether Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1 protein could be correctly expressed, we performed SDS-PAGE and scanning electron microscopy analysis on purified PVC.
Upon analyzing the SDS-PAGE results, we observed a distinct band at approximately 69 kDa, which closely resembles Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1. (Figure 3b). In the meantime, negative-stain transmission electron microscopy on purified PVCs (Figure 3c) showed similar structures to the Cre-carrying PVCs in (Figure 1b), suggesting that the Engineered Mitochondrial Uncoupler Pdp1NTD-EGFP-UCP1 could be correctly loaded into the PVCs. Additionally, by incubating these PVC particles with HEK-293T cells transfected with either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids. we demonstrated these PVC_(EGFP-UCP1)^(EGFR-targeting) particles could selectively enhance the energy expenditure in EGFR-expressing cells (Figure 3d). Altogether, our findings demonstrate a successful engineer of a PVC-based strategy to boost cellular energy expenditure by specifically deliver UCP1 into target cells.