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| | <h2>Results by Section</h2> | | <h2>Results by Section</h2> |
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| − | <a href="https://2018.igem.org/Team:UNSW_Australia/Lab/Cloning">
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| | <div class="overall box flex-center"> | | <div class="overall box flex-center"> |
| | <div class="att-photo"> | | <div class="att-photo"> |
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| | </div> | | </div> |
| | </div> | | </div> |
| − | </a>
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| | | | |
| − | <a href="https://2018.igem.org/Team:UNSW_Australia/Lab/Cloning">
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| − | <div id="cloning" class="subsection box">
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| − | <div class="flex-center">
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| − | <div height="120px" width="120px" class="icon">
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| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/0/0a/T--UNSW_Australia--Icon-cloning.png">
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| − | </div>
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| − | </div>
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| − | <div>
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| − | <h2>Cloning</h2>
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| − | </div>
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| − | <div>
| |
| − | <h3>Summary of Results</h3>
| |
| − | <p>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 to clone the DNA inserts into vectors in one simple isothermal reaction<sup><href="#references">1</sup>. The products of the reaction were transformed into competent DH5-alpha <i>Escherichia coli</i> 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.</p>
| |
| − | <h3>Discussion</h3>
| |
| − | <p>All 8 original DNA constructs were successfully cloned into pETDuet-1 and pRSF-Duet1 plasmid vectors. However, these recombinant plasmids were unable to be expressed. We hypothesise that this was due to the 20 bp long BioBrick prefix situated between the ribosomal binding site (RBS) and the start codon of these construct. This displaces the RBS away from the start of transcription, which is the likely cause for the difficulties experienced with protein expression.</p>
| |
| − | <p>Following these challenges with our pETDuet-1 and pRSFDuet-1 recombinant plasmids, we modified 6 of our DNA constructs and successfully cloned them 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 insertion of our DNA constructs a diagnostic digest was performed as well as Sanger sequencing.</p>
| |
| − | <h3>Future Plans</h3>
| |
| − | <p>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.<p>
| |
| − | </div>
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| − | </div>
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| − | </a>
| |
| | | | |
| − | <a href="https://2018.igem.org/Team:UNSW_Australia/Lab/Protein">
| + | <div class="overall box flex-center"> |
| − | <div id="protein" class="subsection box"> | + | <div class="att-photo"> |
| − | <div class="flex-center">
| + | <img src=https://static.igem.org/mediawiki/2018/c/c7/T--UNSW_Australia--Icon-protein.png> |
| − | <div height="120px" width="120px" class=" icon"> | + | |
| − | <img height="120px" width="120px" src= "https://static.igem.org/mediawiki/2018/c/c7/T--UNSW_Australia--Icon-protein.png "> | + | |
| − | </div>
| + | |
| | </div> | | </div> |
| − | <div> | + | <div class="att-text vertical-align-margin"> |
| − | <h2>Protein Production</h2>
| + | <h3>Protein Production</h3> |
| − | <h3>Summary of Results</h3>
| + | <p>Nine proteins were expressed in <em>Escherichia coli</em> 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.</p> |
| − | <p> Protein scaffold components and proteins that attach to the scaffold needed to be expressed and purified for self-assembly and enzyme activity experiments. Sequence-verified plasmids were heat shock transformed into < i>Escherichia coli</ i> T7 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.</p> | + | |
| − | <h3>Discussion</h3>
| + | |
| − | <p>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 <i>E. coli</i> 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.</p>
| + | |
| − | <p>Despite these difficulties, 9 proteins were successfully purified, and 3 protein constructs from the iGEM Registry were additionally expressed. The following constructs were successfully purified:</p>
| + | |
| − | <ul>
| + | |
| − | <li>His-aPFD & His-bPFD – for assembly of the aPFD/bPFD hexamer, and as a negative control for the effect of scaffolding on enzyme activity.</li>
| + | |
| − | <li>His-aPFD-SpyCatcher & His-bPFD-SnoopCatcher – the scaffold components of our complex that can covalently attach Spy-Tagged and Snoop-Tagged enzymes</li>
| + | |
| − | <li>His-mVenus & His-mCerulean3 – for FRET experiments to investigate the distance between proteins attached to the scaffold.</li>
| + | |
| − | <li>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.</li>
| + | |
| − | <li>His-IaaH-SpyTag – the second enzyme of our reaction pathway, indole-3-acetamide hydrolase, fused with a SpyTag for attachment to His-aPFD-SpyCatcher.</li>
| + | |
| − | </ul>
| + | |
| − | <p>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.</p>
| + | |
| − | <h3>Future Plans</h3>
| + | |
| − | <p>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.</p>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </a>
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| | | | |
| − | <a href=https://2018.igem.org/Team:UNSW_Australia/Lab/Assembly>
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| − | <div id="assembly" class="subsection box">
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| − | <div class="flex-center">
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| − | <div height="120px" width="120px" class="icon">
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| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/c/c4/T--UNSW_Australia--Icon-assembly.png">
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| − | </div>
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| − | </div>
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| − | <div>
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| − | <h2>Assembly</h2>
| |
| − | <h3>Summary of Results</h3>
| |
| − | <p>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. 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. Through TEM, we also visualised wild-type gamma prefoldin, gamma prefoldin fused to SpyCatcher, as well as gamma prefoldin-SpyCatcher attached to IaaH-SpyTag.</p>
| |
| − | <h3>Discussion</h3>
| |
| − | <p>Lorem ipsum</p>
| |
| − | <h3>Future Plans</h3>
| |
| − | <p>Lorem ipsum</p>
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| − | </div>
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| − | </div>
| |
| − | </a>
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| | | | |
| − | <a href=https://2018.igem.org/Team:UNSW_Australia/Lab/FRET>
| + | <div class="overall box flex-center"> |
| − | <div id="fret" class="subsection box"> | + | <div class="att-photo"> |
| − | <div class="flex-center">
| + | <img src=https://static.igem.org/mediawiki/2018/c/c4/T--UNSW_Australia--Icon-assembly.png> |
| − | <div height="120px" width="120px" class="icon"> | + | |
| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/3/34/T--UNSW_Australia--Icon-fret.png"> | + | |
| | </div> | | </div> |
| − | </div> | + | <div class="att-text vertical-align-margin"> |
| − | <div>
| + | <h3>Assembly</h3> |
| − | <h2>FRET</h2>
| + | <p>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.</p> |
| − | <h3>Summary of Results</h3>
| + | |
| − | <p> 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. We started by obtaining purified mCerulean3 and mVenus and performing FRET using these unscaffolded proteins. Due to time constraints, we did not have the opportunity to perform this experiment using scaffolded mCerulean3 and mVenus. Nevertheless, we have established FRET protocols to use in the future, and have obtained the optimum excitation wavelength to use in future FRET experiments with scaffolded mCerulean3 and mVenus.</ p> | + | |
| − | <h3>Discussion</h3>
| + | |
| − | <p>Due to delays in the cloning and protein expression side of the experimentation, the two proteins mCerulean3 and mVenus 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, mCerulean3 and mVenus were expressed from the expression vector pET19b. Instead, mCerulean3 and mVenus were purified after obtaining the expression plasmids from Dr Dominic Glover's laboratory on campus at UNSW. Despite being unable to perform the FRET that was planned for our scaffold, we achieved the first aim of our FRET experiments, generating data on the negative controls for our future FRET experiments with scaffolded mCerulean3 and mVenus, as well as the optimum excitation and emission wavelengths of the fluorescent proteins.</p>
| + | |
| − | <h3>Future Plans</h3>
| + | |
| − | <p>The future directions that FRET would take would be to successfully express the two proteins mCerulean3 and mVenus as fusions with Snoop and Spy Tag so that they may be attached to our Assemblase scaffold as originally planned. This would allow us to measure the distance between the scaffolded molecules and hence more accurately model the system based on experimental data.</p>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </a>
| |
| | | | |
| − | <a href=https://2018.igem.org/Team:UNSW_Australia/Lab/Assays>
| + | |
| − | <div id="assays" class="subsection box"> | + | <div class="overall box flex-center"> |
| − | <div class="flex-center">
| + | <div class="att-photo"> |
| − | <div height="120px" width="120px" class="icon"> | + | <img src=https://static.igem.org/mediawiki/2018/3/34/T--UNSW_Australia--Icon-fret.png> |
| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/e/e1/T--UNSW_Australia--Icon-assay.png"> | + | |
| | </div> | | </div> |
| − | </div> | + | <div class="att-text vertical-align-margin"> |
| − | <div>
| + | <h3>FRET</h3> |
| − | <h2>Enzyme Assays</h2>
| + | <p>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.</p> |
| − | <h3>Summary of Results</h3>
| + | |
| − | <p> Biosynthesis of indole acetic acid (IAA) is the two -step enzymatic reaction we selected to use as proof of concept of our Assemblase scaffold. We developed assays to evaluate the effectiveness of our scaffold in improving IAA yield by quantifying the amount of IAA pathway reactants and products in a reaction over time. This would allow us to compare the quantity of IAA produced by non-scaffolded enzymes versus enzymes co-localised with our Assemblase scaffold . In these experiments we quantified the two reactants, tryptophan and indole acetamide (IAM), alongside the final product 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.</p> | + | |
| − | <h3>Discussion</h3>
| + | |
| − | <p>The Salkowski assay is a suitable and relatively inexpensive tool to determine the concentration of IAA when it is pure in solution, giving a R<sup>2</sup> 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.</p>
| + | |
| − | <p>HPLC analysis produced a far more accurate standard curves for IAA (R<sup>2</sup> = 0.999), and very accurate curves for IAM (R<sup>2</sup> = 1) and tryptophan (R<sup>2</sup> = 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.</p>
| + | |
| − | <h3>Future Plans</h3>
| + | |
| − | <p>Further analysis of the changing concentration of IAA in IaaH and 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 and IaaM to determine the effect of enzyme co-localisation on reaction rate and product yield.</p>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| − | </a>
| |
| | | | |
| − | <a href=https://2018.igem.org/Team:UNSW_Australia/Lab/Plants>
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| − | <div id="plants" class="subsection box">
| |
| − | <div class="flex-center">
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| − | <div height="120px" width="120px" class="icon">
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| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/d/d0/T--UNSW_Australia--Icon-plants.png">
| |
| − | </div>
| |
| − | </div>
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| − | <div>
| |
| − | <h2>Plants</h2>
| |
| − | <h3>Summary of Results</h3>
| |
| − | <p>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. <i>Arabidopsis thaliana</i> 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.</p>
| |
| − | <h3>Discussion</h3>
| |
| − | <p><b>Effect of IAA on <i>Arabidopsis thaliana growth</i></b></p>
| |
| − | <p>The effects of auxins on plant growth and development varies with concentration<sup><href="#references">2,<href="#references">3</sup>. 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 <i>A. thaliana</i><sup><href="#references">4</sup>. At lower concentrations, IAA has been shown to promote root elongation<sup><href="#references">5</sup>, 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 <i>A. thaliana</i><sup><href="#references">6, <href="#references">7</sup>.</p>
| |
| − | <p>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 <i>A. thaliana</i><sup><href="#references">8</sup>.</p>
| |
| − | <p><b>IAA (in)stability</b></p>
| |
| − | <p>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)<sup><href="#references">9</sup> and allows for cultured specimens grown in the laboratory to be transplanted into soil and grown outside.<p/>
| |
| − | <p> 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 media<sup><href="#references">10</sup>. 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 IBA<sup><href="#references">11</sup>. 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.</p>
| |
| − | <h3>Future Plans</h3>
| |
| − | <p>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</p>
| |
| | | | |
| | + | <div class="overall box flex-center"> |
| | + | <div class="att-photo"> |
| | + | <img src=https://static.igem.org/mediawiki/2018/e/e1/T--UNSW_Australia--Icon-assay.png> |
| | </div> | | </div> |
| − | </div> | + | <div class="att-text vertical-align-margin"> |
| − | </a>
| + | <h3>Enzyme assays</h3> |
| − | | + | <p>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. </p> |
| − | <div id="successes" class="subsection box">
| + | |
| − | <div height="120px" width="120px" class="icon">
| + | |
| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/c/ca/T--UNSW_Australia--results-tick.png">
| + | |
| − | </div> | + | |
| − | <div>
| + | |
| − | <h2>Successes</h2>
| + | |
| − | <ul>
| + | |
| − | <li>Eight constructs cloned into corresponding pET-Duet1 and pRSF-Duet1 plasmids</li>
| + | |
| − | <li>Six constructs cloned into pET-19b plasmids</li>
| + | |
| − | <li>Nine proteins expressed and purified</li>
| + | |
| − | <li>Three constructs from the iGEM registry were expressed</li>
| + | |
| − | <li>Demonsrated formation of aPFD and bPFD hexamers</li>
| + | |
| − | <li>Able to covalently attach IaaH-SpyTag to aPFD-SpyCatcher and gPFD-SpyCatcher</li>
| + | |
| − | <li>TEM performed on wild type gPFD, gPFD-SpyC and gPFD-SpyC reacted with IaaH-SpyT</li>
| + | |
| − | <li>FRET with gamma prefoldin and fluorescent tags successful, demonstrating modularity</li>
| + | |
| − | <li>Standards for the Salkowski assay and HPLC using the indole-acetic acid biosynthesis pathway established</li>
| + | |
| − | <li>Protocol developed to observe the functionality of IAA through a plant growth assay, IAA demonstrated to have an effect on plant root growth</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| | | | |
| | | | |
| − | <div id="failures" class="subsection box"> | + | <div class="overall box flex-center"> |
| − | <div height="120px" width="120px" class="icon"> | + | <div class="att-photo"> |
| − | <img height="120px" width="120px" src="https://static.igem.org/mediawiki/2018/9/99/T--UNSW_Australia--results-cross.png"> | + | <img src=https://static.igem.org/mediawiki/2018/d/d0/T--UNSW_Australia--Icon-plants.png> |
| − | </div>
| + | |
| − | <div>
| + | |
| − | <h2>Failures</h2>
| + | |
| − | <ul>
| + | |
| − | <li>Original set of G-Blocks cloned into pETDuet-1 and pRSFDuet-1 failed to express proteins</li>
| + | |
| − | <li>Unable to clone mCerulean3-SnoopTag and mVenus3-SpyTag into pET-19b for FRET experiments due to time constraints</li>
| + | |
| − | <li>Encountered issues with solubility of some proteins</li>
| + | |
| − | <li>Difficulties in quantifying concentrations of IAA over time using the Salkowski assay</li>
| + | |
| − | <li>Unable to conduct plant experiments using IAA synthesised by the protein-enzyme scaffold</li>
| + | |
| − | </ul>
| + | |
| | </div> | | </div> |
| | + | <div class="att-text vertical-align-margin"> |
| | + | <h3>Plants</h3> |
| | + | <p>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.</p> |
| | </div> | | </div> |
| | </div> | | </div> |
| | + | |
| | | | |
| | <div id="references-law"> | | <div id="references-law"> |
