Difference between revisions of "Part:BBa K3089021"

Revision as of 11:07, 16 October 2019

T7 promoter+csgA-linker-mfp5-His fusion protein

This composite parts is meant to express csgA-linker-mfp5 fusion genes. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing mechanical cohesive strength and Mfp5 is a mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. This recombinant protein would self-assemble into fibrous bundles or films with adhesive properties by displaying the mussel adhesion domains on the surface of amyloid scaffolds, which would be a promising new generation of bio-inspired adhesives for a wide range of applications. This part was designed based on the core part——mfp5, which has been submitted into parts registry by iGEM2015 Tu_delft (BBa_K1583002).

Characterization

BBa_K3089021 was characterized in following experiments:

• Protein expression
• Protein purification
• Surface coating analysis

Figure. 2 Geraniol production analysis by gas chromatography. (A) GPPS and GES are arranged in operon regulated by pTac, placed on a high copy vector pUC20. (B) The MVA pathway is split into two clusters and placed on a low copy vector with the upper cluster containing three genes and the downstream containing four. (C) A combined plasmid with GPPS&GES operon and MVA pathway with a low copy p15A origin. (D) Gas chromatography for geraniol produced by E. coli expressing pMVA only (middle trace) or pMVA-GPPS-GES (bottom trace) whose peaks coincide with a geraniol standard (top trace). (E) Geraniol yield is noticeable with only the heterologous MVA pathway. Upon introduction of GPPS and GES, the yield doubled relative to the negative control pMVA only.

Figure. 2 Geraniol production analysis by gas chromatography. (A) GPPS and GES are arranged in operon regulated by pTac, placed on a high copy vector pUC20. (B) The MVA pathway is split into two clusters and placed on a low copy vector with the upper cluster containing three genes and the downstream containing four. (C) A combined plasmid with GPPS&GES operon and MVA pathway with a low copy p15A origin. (D) Gas chromatography for geraniol produced by E. coli expressing pMVA only (middle trace) or pMVA-GPPS-GES (bottom trace) whose peaks coincide with a geraniol standard (top trace). (E) Geraniol yield is noticeable with only the heterologous MVA pathway. Upon introduction of GPPS and GES, the yield doubled relative to the negative control pMVA only.