Coding

Part:BBa_K4960036

Designed by: Ziying Wang   Group: iGEM23_NUDT-CHINA   (2023-10-11)
Revision as of 20:58, 11 October 2023 by Aaachengcheng (Talk | contribs)


pPVC promoter->pvc1-pvc12->pvc13_NTD-1*GGGSG-CKGGRAKDC-1*GGGSG -pvc13_CTD->pvc14-pvc16

Profile

Name: pvc1-pvc12,pvc13_NTD-1*GGGSG-CKGGRAKDC-1*GGGSG -pvc13_CTD,pvc14-pvc16
Base Pairs: 19423bp
Origin: Synthetic
Properties: A flexible linker which can influence the structure of tail fiber connected to the CKGGRAKDC.

Usage and Biology

The system is a syringe-like macromolecular complex that inject protein payloads into fat cells by driving a spike through the cellular membrane.
The system contains a rigid tube structure housed in a contractile sheath. It is anchored to a baseplate and sharpened by a spike protein.
Different payloads can be load into the lumen of the inner tube behind the spike, form fusion proteins with the tube, or associate with the spike itself. After specific recognition and binding to adipocytes, it can cross the via sheath contraction.[1]
The linker 1*GGGSG on both sides of CKGGRAKDC can affect the function and efficiency of bond and recognition by affecting the structure of the tail protein.(Fig.1)

Figure 1.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.

Special Design

Use the CKGGRAKDC to target fat cells.
The different linker(1*GGGSG or 3*GGGSG) on both sides of the CKGGRAKDC can affect the structure and function of the tail protein. By altering the linker on both sides of the CKGGRAKDC, we explored the most suitable structure of the tail protein for recognition and bond. At the same time, because of the existence of linker, we can use Golden Gate to replace the sequences in the middle of linker more efficiently than enzyme cutting and enzyme linking.

Sequence and Features

Tube:pvc1, pvc5, pvc7
Sheath:pvc2, pvc3, pvc4
Sheath terminator:pvc15
Tail f¬bres:pvc13_NTD-1*GGGSG-CKGGRAKDC-1*GGGSG -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 16960
    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 17491
    Illegal NgoMIV site found at 17630
    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 17929
    Illegal AgeI site found at 19617
  • 1000
    COMPATIBLE WITH RFC[1000]


References

[1]Kreitz J, Friedrich MJ, Guru A, Lash B, Saito M, Macrae RK, Zhang F. Programmable protein delivery with a bacterial contractile injection system. Nature. 2023 Apr.

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