Part:BBa_K4960031

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

Profile

Name:Core expression cassette of pPayload plasmid to generate PVCs carrying Pdp1NTD-EGFP-UCP1 Profile
Origin: Photorhabdus, Aequorea Victoria, Homo Sapiens
Base Pairs:4796bp

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 one of these systems containing 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]

This part is the core component of the pPayload plasmid that packs UCP1 into the PVCs. It contains five coding genes that are responsible for payload packaging and the regulating the PVC assembly (Figure.1). 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.

This part, working together with pPVC plasmids encoding the other structural and accessory genes (e.g. BBa_K4960040) would allow the generation of functional PVCs that deliver UCP1 into the target cell, thereby regulating the energy expenditure of the target cells.

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

According to the work of Kreitz et al., The four regulatory genes PAU_RS16570-RS24015 (BBa K4960017-K4960020) are responsible for regulating the assembly of PVC particles. Whether it was possible to remove some of these genes remains elusive. In order to make the PVC system simpler, we tried to remove PAU_RS16570， one of our regulatory genes， from this part. The negative-stain transmission electron microscopy on purified PVCs showed without this gene, PVC cannot be assembled to function properly. (Figure.2) Hence, we decided to keep the complete set of regulatory genes in this part.

Figure 2.Charactrization of the assembled PVC_(EGFP-UCP1)^(EGFR-targeting) ΔPAU_RS16570 particles by negative-strain TEM. TEM images depicting the results of purification without PAU_RS16570, indicating t the four regulatory genes are essential for proper PVC folding. Scale bar, 600 nm.

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.

Functional test

Methods
To validate the function of this part, We constructed pNC093, a pPayload plasmid carrying Pdp1NTD-EGFP-UCP1 payload (BBa_K4960021). 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 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.