Part:BBa_K4960033

pPayload promoter->Pdp1_NDT-3*GGSGG-Cre-GSSG-HiBiT->PAU_RS16570->PAU_RS16565->PAU_RS16560->PAU_RS240

a novel payload loading Cre to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells

Usage and Biology

For the delivery mediated by PVCs,Pdp1 NTD(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’.Therefore,it is rebuilt by the NTD fusion with Cre(BBa K4960023) to verify recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells . PAU_RS24015、PAU_RS16560、PAU_RS16565、 PAU_RS16570(BBa K4960017 BBa K4960018BBa K4960019BBa K4960020) may be involved in gene regulation and possibly contribute to the formation of PVCs in E.coil.
Generally,this part is responsible for producing Cre Payload and loading it into PVC complex in subsequent experiment to confirm previous studies that recombinant PVCs can be reprogrammed to both load and deliver non-native proteins into target cells.

Special Design

Fuse the NTD of Pdp1 with Cre(a novel protein for Payloads)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 910
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 596
  
• 1000
  COMPATIBLE WITH RFC[1000]

Functional Parameters

To demonstrate that Cre could work as our expectation, we transformed E01DARPin (pNC090, similar to the pAWP78-PVCpnf_pvc13-E01DARPin plasmid reported by Kreitz et al.) that specifically targets EGFR and a pPayload plasmid carrying Cre recombinase (pNC091, similar to the pBR322-Pdp1NTD-Cre-HiBiT plasmid reported by Kreitz et al.)into the engineered Escherichia coli.(Fig.1) Then PVCs were purified from E.coli electroporated with pNC090 (pPVC with EGFR targeting E01DARPin) and pNC091 (pPayload with Cre) via ultracentrifuge.(Fig.2) Negative-stain transmission electron microscopy resembled canonical PVC structures similar to the ones reported by Kreitz et al. (Fig.3), suggesting a successful assembly of PVCs.
We incubated these EGFR-targeting, Cre-delivering PVCs with HEK-293T cells co-transfected with pLZ362 (PCMV-LoxP-STOP-LoxP-sNluc, a gift from Lihang Zhang, Westlake University) and either pNC089 (PCMV-EGFR) or pcDNA3.1(+) plasmids.Secreted Nanoluc (sNluc) levels in the culture medium were then evaluated at 72 h post PVCs treatment. Results showed a significant increase in sNluc levels in EGFR-expressing cells treated with PVCs, while the cells without EGFR expression showed low sNluc levels.These results demonstrated that Cre is correct and engineered PVCs could specifically deliver payload proteins into target mammalian cells, thereby serving as a possible delivery mechanism for our purposes.(Fig.4)
Figure 1

Figure 2

Figure 3

Figure 4

Figure 1.the Transformation of Cre and Darpin
Figure 2.Purification
Figure 3.PVC Structures under Electron Microscope
Figure 4.Result