Part:BBa_K3187013
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 | 3556 |
| 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 pTeTW3con2 as backbone and 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.
- 10INCOMPATIBLE 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 - 12INCOMPATIBLE 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 - 21INCOMPATIBLE 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 - 23INCOMPATIBLE 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 - 25INCOMPATIBLE 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 - 1000COMPATIBLE WITH RFC[1000]
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