Coding

Part:BBa_K4960034

Designed by: Ziying Wang   Group: iGEM23_NUDT-CHINA   (2023-10-11)


pPVC promoter->pvc1-pvc12->pvc13_NTD-2*Bsal-pVc13_CTD->pvc14-pvc16

Structural and accessory genes

Profile

Name:pvc1-pvc12,pvc13_NTD-2*Bsal-pVc13_CTD,pvc14-pvc16
Base Pairs: 19822bp
Origin: Photorhabdus
Properties: Structural and accessory genes


Usage and Biology

This part is necessary for the assembly of a functional injection system. PVCs probably recognize target cells via tail fibres (Pvc13), leading to a contraction of the sheath mechanism that drives a spike through the cellular membrane.[1]
Pvc2, Pvc3 and Pvc4 compose the sheath of PVC. Pvc16 is sheath terminator of PVC. Pvc13 is tail fibres of PVC. Pvc9, Pvc11 and Pvc12 compose the baseplate of PVC. Pvc8 and Pvc10 compose the spike of PVC. Pvc1, Pvc5 and Pvc7 compose the tube of PVC.[2]（Fig.1)
This part has a Bsal enzyme recognizing sequence, which can allow to use the way Golden Gate Assembly to construct a plasmid.(Fig.2)

Figure 1.PVC composition diagram

Figure 2 AlphaFold2-guided Design of Adipose Cell-targeting PVC Coat Expressing Plasmids. (a) AlphaFold2 based prediction of engineered PVC tail fiber trimer structure. Structure of adipose-targeting CKGGRAKDC peptide-presenting PVC tail fiber with indicated linkers were shown. (b) Schematic diagram of the newly-designed pvc13 part allowing Golden Gate cloning of targeted sequence into the pPVC plasmid.

Special Design

To enable the PVC-based delivery of UCP1 into white adipose tissue, we then decided to equip the tail fiber protein of PVC (PVC13) with an adipose tissue-targeting 9-mer peptide developed by Kolonin et al. in 2004[3]. To ensure the efficient exposure of the targeting peptide on the tail fiber, we predicted the structure of PVC13 trimers inserted with adipose tissue targeting peptide flanked by different linkers (Figure 2a). We were unable to peruse validating these constructs due to the limited time before the jamboree and the practical difficulties in working with such a huge plasmid (~25kb), while we did make some effort in simplifying the cloning process by designing a new PVC13 part which allows subsequent Golden-gate-based cloning of the targeting sequence into the pPVC plasmid (Figure 2b, see the second part of our Engineering page for more information).

Sequence and Features

Tube:pvc1, pvc5, pvc7
Sheath:pvc2, pvc3, pvc4
Sheath terminator:pvc15
Tail fbres:pvc13_NTD-2*Bsal-pVc13_CTD
Baseplate:pvc9, pvc11, pvc12
Spike:pvc8, pvc10
Assembly method:3A Assembly


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7406
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2407
    Illegal BglII site found at 11264
    Illegal BglII site found at 14099
    Illegal BglII site found at 16930
    Illegal XhoI site found at 2691
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 11792
    Illegal NgoMIV site found at 17461
    Illegal NgoMIV site found at 17600
    Illegal AgeI site found at 6704
    Illegal AgeI site found at 9641
    Illegal AgeI site found at 10327
    Illegal AgeI site found at 10468
    Illegal AgeI site found at 11576
    Illegal AgeI site found at 17899
    Illegal AgeI site found at 19587
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1] J. Kreitz et al., “Programmable protein delivery with a bacterial contractile injection system,” Nature, vol. 616, no. 7956, pp. 357–364, Apr. 2023.
[2] F. Jiang et al., “Cryo-EM Structure and Assembly of an Extracellular Contractile Injection System,” Cell, vol. 177, no. 2, pp. 370-383.e15, Apr. 2019,.
[3]Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R., & Arap, W. (2004). Reversal of obesity by targeted ablation of adipose tissue. Nat Med, 10(6), 625-632. https://doi.org/10.1038/nm1048

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