Difference between revisions of "Team:UNSW Australia/Lab"
Patricia S (Talk | contribs) |
TobiasGaitt (Talk | contribs) |
||
| Line 101: | Line 101: | ||
<div> | <div> | ||
<h2>Plants</h2> | <h2>Plants</h2> | ||
| − | <p>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 <i>Arabidopsis thaliana</i>. The results of the plant growth assays demonstrated that the addition of IAA | + | <p>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 <i>Arabidopsis thaliana</i>. 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.</p> |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 14:29, 16 October 2018
Lab Overview
..
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
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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
Lorem ipsum dolor sit amet, consectetur adipisicing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat. Duis aute irure dolor in reprehenderit in voluptate velit esse cillum dolore eu fugiat nulla pariatur. Excepteur sint occaecat cupidatat non proident, sunt in culpa qui officia deserunt mollit anim id est laborum.
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.