Part:BBa_K4960031

Core expression cassette of pPayload plasmid to generate PVCs carrying Pdp1NTD-EGFP-UCP1

Profile

Name:Pdp1NTD-3*GGSGG-EGFP-2*GGGSG-UCP1->PAU_RS24015->PAU_RS16560->PAU_RS16565->PAU_RS16570
Origin: Photorhabdus, Aequorea Victoria, Homo Sapiens

Usage and Biology

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). Because it has been shown that it can be targeted by synthetic biology methods other than the original insect cells, such as mice and human cells. [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. This part is based on the payload carried by the PVCs system and the gene responsible for regulating the assembly of PVCs (Fig.1). Pdp1NTD-3*GGSGG-EGFP-2*GGGSG-UCP1 (BBa K4960021) is loaded into the PVC system as a payload protein, PAU RS24015 (BBa K4960017), PAU RS16560 (BBa K4960018), PAU RS16565 (BBa K4960019), PAU RS16570 (BBa K4960020) Four regulatory genes play an integral role in the assembly of PVC systems.
For the delivery mediated by PVCs, Pdp1NTD (the N-terminal domain (NTD) of Pdp1) (BBa K4960016) plays a significant role in loading diverse payloads (including the wild type and novel type) into the PVC complex as a ‘packaging domain’ [2].
Based on the composition of this part, it functions as a complete payload and regulatory genes to be packaged into PVC, and finally, the fusion protein is successfully targeted and delivered into the Hek293T cell line and correctly plays the role of promoting glucose metabolism.

Figure 1. Schematic of the PVCpnf locus. It contains 16 structural and accessory genes , followed by two payload genes (Pdp1 and Pnf, in red) and four putative regulatory genes (in pink).

Special Design

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. (Fig.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.

Figure 2. Charactrization of the assembled PVC_(EGFP-UCP1)^(EGFR-targeting) Δ particles by negative-strain TEM

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1569
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 546
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Experimental Validation

This part is validated through functional test.

Method

To validate the function of this part, We constructed pNC093, a pPayload plasmid carrying Pdp1NTD-EGFP-UCP1 payload (BBa). 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 (Fig.3a). To validate whether Pdp1NTD-EGFP-UCP1 protein could be correctly expressed, we performed SDS-PAGE and scanning electron microscopy analysis on purified PVC.

Results

Upon analyzing the SDS-PAGE results, we observed a distinct band at approximately 69 kDa, which closely resembles 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 Pdp1NTD-EGFP-UCP1 protein 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.

Figure 3. Delivery of the Fat Burning Payload through 〖PVC〗_(EGFP-UCP1)^(EGFR-targeting) Particles to HEK-293T Cells.(a) Schematic representation of the construction of PVC_(EGFP-UCP1)^(EGFR-targeting) particles. (b, c) Charactrization of the assembled PVC_(EGFP-UCP1)^(EGFR-targeting) particles by SDS-PAGE (b) and negative-strain TEM (c). Scale bars, 100nm. (d) Charactrization of cellular metabolism in PVC_(EGFP-UCP1)^(EGFR-targeting) treated HEK-293T cells transfected with either pNC089 or pcDNA3.1(+) plasmids. Glucose concentration in the cell culture medium was measured 48 h after PVC_(EGFP-UCP1)^(EGFR-targeting) administration; data shows mean±SD, n=3 independent experiments.

References

[1] Kreitz, J., Friedrich, M.J., Guru, A. et al. Programmable protein delivery with a bacterial contractile injection system. Nature 616, 357–364 (2023).
[2] Jiang F, Shen J, Cheng J, Wang X, Yang J, Li N, Gao N, Jin Q. N-terminal signal peptides facilitate the engineering of PVC complex as a potent protein delivery system. Sci Adv. 2022 Apr 29;8(17):eabm2343.