Composite

Part:BBa_K3187012

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-12)

pTet sfGFP-P22 Bacteriophage Scaffolding Protein x Coat Protein pT7 (Convergent)

Profile

Name pTeTW3con2-ptet-sfGFP-StrepTag-SP—CP+LPETGG-pT7
Base pairs 2951
Molecular weight 46.1 kDa (sfGFP – StrepTagII – scaffold protein) + 48.9 kDa (coat protein - StrepTagII - LPETGG)
Origin Synthetic
Parts tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, coat protein, scaffold protein, sfGFP, LPETGG, StrepTagII
Properties Production of P22 Virus-like particles with a modifiable surface and sfGFP as cargo.


Usage and Biology

This composite part is a dual expression plasmid with pTeTW3con2 as backbone and two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site (BBa_K3187043) encodes the fusion protein of sfGFP, Strep-Tag and scaffold protein. The T7 promoter can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the site (BBa_K3187044 ) encodes the fusion protein of coat protein, Strep-Tag and LPETGG. The composite part can therefore be used for the production of P22 Virus-like particles (VLPs), as they consist of coat and scaffold protein.

The scaffold fusion protein also includes a Strep-Tag for purification, as well as sfGFP. As a result of the fusion of sfGFP with the scaffold protein the VLPs will contain sfGFP as a cargo on the inside.

The coat fusion protein also includes a Strep-Tag (BBa_K3187025 ) for purification and a LPETGG tag (BBa_K3187019 ). As a result of the fusion of the LEPTGG tag with the coat protein the VLPs will present the tag on the outside. This tag is the recognition sequence of Sortase A7M (BBa_K3187028 ) which catalyzes a covalent bond of the LPETGG and a poly G tag. On this basis the VLPs can be modified on the outside using SortaseA7M after the assembly of the coat and scaffold proteins.

The expression levels of both sites were characterized using the BBa_K3187011.




Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 2900
    Illegal PstI site found at 1334
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 76
    Illegal NheI site found at 1339
    Illegal PstI site found at 1334
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 76
    Illegal BamHI site found at 796
    Illegal XhoI site found at 1499
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 2900
    Illegal PstI site found at 1334
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 76
    Illegal XbaI site found at 2900
    Illegal PstI site found at 1334
    Illegal NgoMIV site found at 1185
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 94


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