Team:UNSW Australia/Lab

Lab Sections

DNA Cloning

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.

Protein Production

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..

Assembly

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 hexameric structures, attach the IaaH-SpyTag protein to alpha prefoldin and gamma prefoldin scaffolds and visualise SpyCatcher-SpyTag interaction.

FRET

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.

Enzyme Assays

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Plants

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.