Composite

Part:BBa_K3187013

Designed by: iGEM TU_Darmstadt 2019   Group: iGEM19_TU_Darmstadt   (2019-10-13)
Revision as of 14:22, 17 October 2019 by ToPro (Talk | contribs)


P22 Coat and Scaffolding Protein x Coat Protein with LPETGG-tag for sortase-mediated ligation

Profile

Name pTeTW3con2-ptet-SP-CP--CP-LPETGG-pT7
Base pairs 3548
Molecular weight 46.9 kDa (coat protein) + 18 kDa (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, LPETGG, StrepTagII
Properties Production of P22 Virus-like particles with an adjustable level of modification on the surface.

Usage and Biology

This composite part is a dual expression plasmid with two convergent expression sites. The tetA promoter is inducible with anhydrotetracycline (AHT) and the site (BBa_K3187045) encodes the coat 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, StrepTagII and LPETGG. It was cloned to produce P22 Virus-like particles (VLPs) which consist of coat and scaffold proteins.

The fusion protein which includes the coat protein, also includes a StepTagII (BBa_K3187025) for purification and a LPETGG tag. The assembled VLPs will present the LPETGG tag on the outside as a result of this design. LPETGG (BBa_K3187019) is the recognition sequence of Sortase A7M (BBa_K3187028) which catalyzes a covalent bonding of the LPETGG and a poly G tag. The poly G tag can be fused to the N-terminus and the LPETGG tag to the C-terminus of a protein of interest. On this basis the VLPs can be modified on the outside using the Sortase A7M after the assembly of the coat and scaffold protein.

The adjustable level of modification can be reached by tuning the expression of the two sites, e.g. using different concentrations of the inducers. This leads to a change in ratio of LPETGG-tagged and tagless coat protein. 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 EcoRI site found at 1395
    Illegal XbaI site found at 3505
    Illegal PstI site found at 1931
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 76
    Illegal EcoRI site found at 1395
    Illegal NheI site found at 1936
    Illegal PstI site found at 1931
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 76
    Illegal EcoRI site found at 1395
    Illegal BamHI site found at 1389
    Illegal XhoI site found at 2104
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 76
    Illegal EcoRI site found at 1395
    Illegal XbaI site found at 3505
    Illegal PstI site found at 1931
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 76
    Illegal EcoRI site found at 1395
    Illegal XbaI site found at 3505
    Illegal PstI site found at 1931
    Illegal NgoMIV site found at 1783
  • 1000
    COMPATIBLE WITH RFC[1000]


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