Cloning

Summary of Results

To express the various components of our scaffold for protein conjugation and assembly experiments, our DNA constructs were cloned into appropriate plasmid vectors. We used Gibson assembly cloning to assemble the DNA inserts with the vectors in one simple isothermal reaction¹. The products of the reaction were transformed into competent DH5-alpha Escherichia coli cells and colonies were screened for recombinant plasmids. Plasmids containing the desired DNA inserts were transformed into T7 cells for protein expression and purification experiments. In total, eight constructs were successfully cloned into corresponding pET-Duet1 and pRSF-Duet1 plasmids, while 6 constructs were cloned into pET-19b plasmids.

Discussion

All 8 original DNA constructs were successfully cloned into pETDuet-1 and pRSF-Duet1 plasmid vectors, and 6 DNA constructs were successfully cloned into pET-19b vectors. In addition to this, BBa_K515000 and BBa_K515001 parts were also cloned into pET-19b for BioBrick experiments. To confirm the successful insert of our DNA constructs, the plasmids were digested with restriction enzymes and products were analysed with agarose gel electrophoresis.

Recombinant pETDuet-1 and pRSFDuet-1 plasmids were unable to be expressed and it was hypothesised that this was due to the 20 bp long BioBrick prefix situated between the ribosomal binding site (RBS) and the start codon of the construct. This displaces the RBS away from the start of transcription, which is the likely cause for the difficulties experienced with protein expression.

Future Plans

The DNA constructs were cloned into the pET-19b vector as it was used successfully in previous cloning experiments for our collaborators. In comparison to the previous Duet vectors, we were able to achieve successful protein expression with the recombinant pET-19b plasmids. In the future, we hope to clone more parts into suitable vectors, including the enzymes that are involved in the taxol synthesis pathway. This would enable us to perform further assembly tests with different enzymes, and to ultimately piece together and characterise a range of functional scaffolds.

Protein Production

Summary of Results

Protein scaffold components and proteins that attach to the scaffold must be expressed and purified for self-assembly and enzyme activity experiments. Sequence-verified plasmids were heat shock transformed into Escherichia coli cells and expressed for recombinant protein production. The proteins were then purified from cell lysates with Immobilised Metal Affinity Chromatography (IMAC) columns. Nine proteins have been successfully expressed and purified, enabling the construction of our scaffold-enzyme complex.

Discussion

Initial attempts at protein expression were unsuccessful using pET-Duet-1 and pRSF-Duet-1 plasmids. After cloning our desired inserts into these plasmids, we attempted to express these proteins, but no expression could be detected by SDS-PAGE or Western Blot. Both E. coli T7 Express and Lemo21(DE3) cell lines were used for expression, and tested with 0.1 mM, 0.4 mM and 1 mM IPTG inductions. We hypothesised that the design of our plasmids inhibited expression, as the BioBrick prefix was placed between the ribosome binding site and the start codon of our coding sequence. We decided to subclone our inserts into pET-19b and remove the BioBrick prefix and suffix before retrying protein expression and purification.

Despite these difficulties, 9 proteins were successfully purified, and 3 protein constructs from the iGEM Registry were expressed. The following constructs were successfully purified:

• His-aPFD & His-bPFD – for assembly of the aPFD/bPFD hexamer, and as a negative control for the effect of scaffolding on enzyme activity.
• His-aPFD-SpyCatcher & His-bPFD-SnoopCatcher – the scaffold components of our complex that can covalently attach Spy-Tagged and Snoop-Tagged enzymes
• His-mVenus & His-mCerulean3 – for FRET experiments to investigate the distance between proteins attached to the scaffold.
• His-gPFD-SpyCatcher & His-SpyCatcher-gPFD-SpyCatcher – filamentous variants of prefoldin fused with SpyCatchers, to test SpyTag/SpyCatcher reactions and to determine if gPFD can form filaments with enzymes attached to its N- and/or C- terminus.
• His-IaaH-SpyTag – the second enzyme of our reaction pathway, indole-3-acetamide hydrolase, fused with a SpyTag for attachment to His-aPFD-SpyCatcher.

Following protein purification, we encountered issues with solubility for some proteins. This was likely due to non-optimal buffer conditions or high concentrations of protein. Further experimentation and optimisation is required to identify the range of conditions in which these proteins are stable and able to assemble.

Future Plans

In the future, we would like to express and purify all of our successfully cloned constructs, including the first enzyme of our reaction pathway, tryptophan-2-monooxygenase (IaaM), fused to the SnoopTag, fluorescent proteins with appropriate tags for FRET experiments and enzymes from other pathways fused with SpyTag and SnoopTag. In addition, we would like to increase the purity of our purifications and attempt larger scale protein expressions. These provide materials that are fundamental for the characterisation of the assembly of our scaffold, the distance between the attachment site, the rate of indole acetic acid production and the modularity of our system.

Assembly

Summary of Results

The formation of our enzyme-scaffold complex requires two stages of assembly – the formation of the alpha prefoldin and beta prefoldin hexamer and the covalent attachment of enzymes to the scaffold through SpyTag/SpyCatcher or SnoopTag/SnoopCatcher reactions. These assembly stages were characterised separately, by Size Exclusion Chromatography (SEC) and SDS-PAGE respectively. We have demonstrated the formation of alpha and beta prefoldin hexameric and other oligomeric structures, successfully covalently attached the IaaH-SpyTag protein to alpha prefoldin and gamma prefoldin scaffolds and visualised wild type gamma prefoldin, gamma prefoldin fused to SpyCatcher and gamma prefoldin fused to SpyCatcher and IaaH-SpyTag filaments.

Discussion

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Future Plans

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FRET

Summary of Results

It was hoped that the two fluorescent proteins mCerulean3 (Cerulean) and mVenus (Venus) could be expressed as fusion proteins with SnoopTag and SpyTag respectively so that they may be attached to our scaffold. The use of fluorescent proteins allows us to not only show the modularity of our system, but also means we can perform experiments such as FRET to show modularity, as well as measure the distance between the attached fluorophores. However, due to protein expression issues, the molecules tested were the gamma prefoldin and fluorescent tag negative controls from Dr. Donald Glover's lab, and these two did emit fluroscence.

Discussion

Due to delays in the cloning and protein expression side of the experimentation, the two proteins Cerulean and Venus were not expressed as fusions to Snoop and Spy Catcher. This means that they could not be scaffolded and FRET was not performed on the scaffolded molecules. Instead, Cerulean and Venus were expressed from the expression vector pET19b. These versions were only tagged with 6 histidine residues for purification. Despite not being able to perform the FRET that was planned for our scaffold, these two that were successfully expressed and purified allowed us to generate data on the negative controls of the experiment, as well as the optimum excitation and emission wavelengths of the fluorescent molecules.

One of the limitations of FRET is the overlap of curves seen between any two fluorophores. The excitation and emission spectra must be close enough so that the fluorophores interact, yet sufficiently distant to limit signal generated in the second fluorophore from the excitation wavelength meant for the first molecule. A balance between these is sought, yet no pairing of fluorophores is perfect, and hence there will always be some level of fluorescence observed in a solution where FRET is not occurring, which was observed in our data.

Future Plans

The future directions that FRET would take would be to successfully express the two proteins Cerulean and Venus as fusions to Snoop and Spy Tag so that they may be scaffolded on to our prefoldin molecule as planned. This would allow us to measure the distance between the molecules that we would be scaffolding and hence more accurately model the system based on experimental data.

Enzyme Assays

Summary of Lab

Assays were used to determine the presence of the reactants and products in the Indole Acetic Acid (IAA) production pathway. The 3 reactants tested were tryptophan, Indole Acetamide (IAM) , and IAA. We used two different assays, the Salkowski assay adapted from the 2011 Imperial College London team, and HPLC. The Salkowski assay is a simple, fast, and inexpensive method of determining the amount of the final product IAA produced over a given time period, and HPLC gathers more detailed data about the relative abundance of each of the reactants and products over a given time period. Only standards were successfully run.

Discussion

The Salkowski assay is a suitable and relatively inexpensive tool to determine the concentration of IAA when it is pure in solution, giving a R² of 0.994 for the initial IAA concentration determination. Problems arise when the assay is used to quantify IAA in the presence of IAM, as due to high absorbance observed when the Salkowski reagent is incubated with IAM; it is likely it the reagent also emits light at 530nm upon reacting with IAM. The Salkowski assay also appeared ineffective in differentiating concentrations of IAA in cell based assays, and due to its nature, it is unable to quantify changes in IAA concentration in a solution over time. These issues limit the Salkowski assay as a basic quantifier of IAA, thus it should only be used as a preliminary step to verify hypotheses about the production of IAA before more costly, effective and accurate methods such as HPLC are employed.

HPLC analysis produced a far more accurate standard curves for IAA (R² = 0.999), and very accurate curves for IAM (R² = 1) and tryptophan (R² = 1). HPLC allowed analysis of all three intermediates with one analysis, and has the potential to measure a changing concentration of these intermediates. Given the high quality and accuracy of standard results produced in the initial HPLC standards, more accurate analysis would provide little benefit as an assay for IAA.

Future Plans

Further analysis of the changing concentration of IAA in IaaH/IaaM expressing cells incubated with tryptophan by HPLC could verify the potential of the enzymes to produce IAA from tryptophan. HPLC could then be used again, on scaffolded IaaH/IaaM incubated in the same conditions to determine any effect scaffolding these enzymes has on changing the rate of overall reaction in the IAA synthesis pathway.

Plants

Summary of Results

Auxins are plant hormones which are 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 protocol was developed to investigate the functionality of biosynthetically produced IAA in a plant growth assay compared to commercially available IAA. Arabidopsis thaliana seedlings were grown in media containing varying concentrations of IAA, to observe its effect on growth and development. Reduced primary root growth and high lateral root number was observed at higher concentrations of IAA, exhibiting phenotypes common with a stress induced morphology. These results, in addition to comments made by PlantBank researchers who had found that addition of exogenous IAA showed no benefit, indicated that IAA synthesis was not an appropriate pathway to purse for commercialisation with our scaffold. Despite this, the UNSW iGEM team was able to develop a protocol that could be used in the future to observe the functionality of the IAA product produced with our scaffold compared with commercially available IAA.

Discussion

Effect of IAA on Arabidopsis thaliana growth

The effects of auxins on plant growth and development varies with concentration^2,3. At high concentration auxins can have an inhibitory effect on cell elongation1, possibly due to the IAA-induced ethylene production, as ethylene has previously been shown to inhibit root elongation in A. thaliana⁴. At lower concentrations, IAA has been shown to promote root elongation⁵, however this effect was not observed in our results. The promotion of lateral root formation at high concentrations of IAA is consistent with previous literature which has shown that IAA promotes, and is essential for lateral root development in A. thaliana^{6, 7}.

Overall, the control specimens that were grown with no IAA appeared to exhibit the best development, with consistently greater leaf and shoot growth, and the plants in the highest concentrations (100 µM IAA) exhibited decreased root elongation and increased formation of lateral roots similar to a stress-induced morphogenic response of A. thaliana⁸.

IAA (in)stability

Our results indicate that exogenous IAA did not facilitate root growth, consistent with comments made by researchers at the PlantBank facilities, with whom we consulted. Auxins are typically used by researchers to stimulate adventitious root growth in their tissue culture specimens, refering to roots which have developed from an unusual location (i.e. leaves or shoots)⁹ and allows for cultured specimens grown in the laboratory to be transplanted into soil and grown outside.

These reachers instead use indole-3-butyric acid (IBA), another auxin, as they had found in previous experiments that IAA showed no benefits and that IBA was easier to work with. These findings may be partially explained by the relative instability of IAA when compared to IBA. Indole-3-aecetic acid is sensitive to photodegradation, which can be accelerated by the minerals present in MS media¹⁰. Although IBA is also subject to photodegradation, the effects are less than that of IAA with tests conducted in MS media finding concentrations of IAA were reduced by more than 97% after 20 days in the light compared to a 60% reduction in IBA¹¹. To account for light sensitivity of IAA, our experimental design was adapted to minimise IAA photodegradation. We conducted all media preparation and plating in a dark laminar flow hood and partially covered our samples.

Future Plans

Due to time constraints, we were not able to conduct experiments with IAA synthesised using our scaffold. However, testing the effects of IAA purchased from Sigma Aldrich allowed us to begin developing a protocol which can be further refined into the future. Primary root growth decreased with increasing IAA concentration which may indicate that the concentrations of IAA used were too high, as reports in the literature state that too much exogenous IAA can inhibit root elongation. Therefore, if we are to re-attempt this experiment in the future we will first have to determine the optimal concentration of IAA to use for our plants