Team:UNSW Australia/Results

Results

Key Findings

Our project has provided the proof of principle for a prefoldin scaffold that co-localises enzymes. We have proven that our attachment mechanisms (SpyTag/Catcher and Snoop Tag/Catcher) work between enzymes and prefoldin, and that the heterohexameric structure of alpha and beta prefoldin self-assembles.

Results by Section

Cloning

Eight DNA constructs were cloned into pETDuet-1 and pRSFDuet-1 plasmids using Gibson assembly. Six of these constructs were subcloned into pET19b for protein expression. In addition to this, BBa_K1789000 (IaaM) and BBa_K1789001 (IaaH) were cloned into pET19b for protein expression. All recombinant plasmids were validated by a diagnostic digest and Sanger sequencing.

Protein Production

Nine proteins were expressed in Escherichia coli T7 cells and purified using Immobilised Metal Affinity Chromatography (IMAC). These included prefoldin proteins that form the basis of our scaffold, and prefoldin proteins fused with SpyCatcher and SnoopCatcher. Enzymes and fluorescent proteins were expressed and purified, including the enzyme indole acetamide hydrolase (IaaH) with a C-terminal SpyTag for conjugation experiments.

Assembly

The formation of our enzyme-scaffold complex requires two stages of assembly: firstly, the formation of the alpha prefoldin and beta prefoldin hexamer, and secondly the covalent attachment of enzymes to the scaffold through SpyTag/SpyCatcher or SnoopTag/SnoopCatcher reactions. We have assembled alpha and beta prefoldin hexamers and covalently attached the enzyme IaaH-SpyTag to alpha prefoldin and gamma prefoldin scaffolds. Furthermore, we visualised filaments of wild-type gamma prefoldin, gamma prefoldin-SpyCatcher, and gamma prefoldin-SpyCatcher attached to IaaH-SpyTag.

FRET

FRET is a method for assessing the distance between two molecules based on energy transfer between two fluorophores. To assess the distance between enzymes attached to our scaffold we planned to perform FRET using the fluorescent proteins mCerulean3 and mVenus free. FRET was successfully performed for mCerulean3 and mVenus free in solution. However, due to time constraints we were unable to express and purify mCerulean3 or mVenus fused to Spy/Snoop Tags for attachment to our scaffold and use in FRET. Nevertheless, we have obtained the optimum excitation wavelengths for mCerulean3 and mVenus and established a working FRET protocol for future use.

Enzyme assays

Biosynthesis of indole-3-acetic-acid (IAA) is the two-step enzymatic reaction we selected to use as proof of concept of our Assemblase scaffold. To compare reaction efficiency in a scaffolded versus unscaffolded enzyme scenario, we successfully set-up two quantitative assays for determining the concentration of reaction intermediates and products over time. We created standard curves for these assays based on unscaffolded enzyme reactions only due to time constraints. In the future these assays can be utilised to test the effect of enzyme scaffolding to our Assemblase system on protein yield.

Plants

Auxins are plant hormones involved in the regulation of plant growth and development. The biosynthesis of the auxin indole-3-aecetic acid (IAA) was used as a test pathway for our scaffold system, and thus a plant growth assay was developed to investigate the functionality of biosynthetically produced IAA. We successfully observed effects of commercially obtained IAA on plant root growth, namely increased lateral root growth and decreased primary root growth. These results are consistent with reports in the literature. Future work could further optimise the concentration of exogenous IAA added to the plant growth agar, and incorporate IAA produced through our Assemblase system.

Enzyme kinetics modelling

The effect of the distance between the enzymes on the reaction rate was modelled. We found that the model predicted a 5-fold improvement in IAA production when the enzymes are clustered together using Prefoldin, compared to when they are free in solution.

Molecular Dynamics

Allowed us to visualise the physical movements of our scaffold and determine key parameters for use in the mathematical model as we were able to approximate the average distance between enzymes when attached to the Prefoldin scaffold.

Software

This tool was developed to provide future iGEM teams an improved tool to scan the Biobricks Database using Python 3. Along with the search tool, we provide a method for generating a clean xml file of the iGEM database as well as incorporating negative searching into the tool to allow for searching of parts that do not meet search criteria.

References

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