Difference between revisions of "Part:BBa K3187013"

Revision as of 13:13, 17 October 2019

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

• Name: pTeTW3con2-ptet-CP-SP—CP+LPETGG-pT7
• Size [bp]: 3548
• Size [kDa]: 46.9 (Coat Protein) + 18 (Scaffold Protein) + 48.9 (Coat Protein - Strep-Tag - LPETGG)
• Origin: Synthetic
• Components: tetA promoter, T7 promoter, T7 terminator, Lac Operator, RBS, Coat Protein, Scaffold Protein, LPETGG, Strep-Tag II
• Feature: Production of P22 Virus-like particles with an adjustable level of modification on the surface

Introduction:

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.

Sequence and Features