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

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).

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

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

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.