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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

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FRET

The expression of fluorescent proteins would enable us to perform FRET experiments and to measure the distance between the attached fluorophores, demonstrating the modularity of the scaffolded system. To achieve this aim, we hoped to express two fluorescent proteins, mCerulean3 (Cerulean) and mVenus (Venus), fused to SnoopTag and SpyTag respectively.

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 IAA on the growth of Arabidopsis thaliana. The results of the plant growth assays demonstrated that the addition of IAA did not benefit root growth, indicating that IAA synthesis was not an appropriate pathway to pursue for commercialisation with our scaffold. Despite this, we were able to develop a protocol that could be used in the future to compare IAA production from our scaffold with commercially available IAA.