To express the various components of our scaffold for self-assembly experiments, our DNA constructs were cloned into plasmid vectors using Gibson assembly cloning methods. The products of the reaction were transformed into competent cells and colonies were screened for recombinant plasmids. 8 constructs were successfully cloned into corresponding pETDuet-1 and pRSFDuet-1 plasmids, while 6 constructs were cloned into pET-19b plasmids.
Components of the protein-enzyme scaffold must be expressed and purified for self-assembly and enzyme activity tests. Sequence-verified plasmids were transformed into Escherichia coli cells and expressed for recombinant protein production. 9 proteins were successfully expressed and purified, enabling the construction of our scaffold-enzyme complex.
To create the enzyme-scaffold complex, the alpha and beta prefoldin hexamer are to be covalently attached to the enzymes through SpyTag/SpyCatcher or SnoopTag/SnoopCatcher reactions. These assembly stages were characterised successfully, and we were able to demonstrate the formation of alpha and beta prefoldin hexamers, covalently attach the IaaH-SpyTag protein to alpha prefoldin and gamma prefoldin scaffolds and visualise filaments of gamma prefoldin attached to enzymes with Transmission Electron Microscopy.
By performing FRET, we aimed to investigate the distance between two fluorescent proteins, mCerulean3 and mVenus, attached to our Assemblase scaffold. This would allow us to gain an understanding of the proximity of enzymes in our Assemblase system and therefore help us perform more accurate modelling of reaction kinetics.
The Salkowski assay and HPLC were used to determine the presence of tryptophan, Indole Acetamide and Indole Acetic Acid (IAA) in the IAA production pathway. The Salkowski assay is a simple, fast and inexpensive method of determining the production of IAA over a specific period of time. The methods utilised in our experiments were adapted from the 2011 Imperial College London team. HPLC experiments enabled us to gather detailed data about the relative abundance of the reactants and products over time.
To examine the functionality of our scaffolded system, the biosynthesis of the auxin indole-3-aecetic acid (IAA) was tested. A protocol was developed to investigate the effect of varying concentrations of commercially available IAA on the growth of Arabidopsis thaliana. The results of the plant growth assays demonstrated that the addition of IAA increased lateral root sprouting whilst inhibiting primary root elongation. We have thus created a protocol which can be used in the future to compare the effect of commercial IAA on plant growth with IAA biosynthesised with our Assemblase scaffolding system.