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Team:Imperial College London/Project/Auxin/Overview
From 2011.igem.org
Indole-3 acetic acid (IAA) is one of the most well studied phytohormones and is also known more commonly under the name Auxin. IAA is known as a key player in the regulation of plant growth and is also a known morphogen implicated in a vast array of processes ranging from embryo patterning to isodiametric expansion (fruit growth).
However, the topic of auxin producing soil bacteria in the rhizosphere has been given little attention so far. We believe that plant-microbe interactions mediated through IAA could be tapped into to modulate the plasticity of the root architecture. In this module, we will be attempting to express Tryptophan monooxygenase (IaaM) and Idoleacetimide hydrolase (IaaH) in Escherichia coli. We are aware that E. coli would not be a suitable chassis for field work and we have taken this into account when we made our DNA sequences.
Design:
IaaM and IaaH have been codon optimized for both Bacillus subtilis and E. coli through the use of our own codon optimizing software. Also, the genes have been placed under the pVEG promoter which works in B. subtilis and E. coli and we calculated the RBS efficiency for both E. coli and B. subtilis. Furthermore, insulator sequences have been placed in front of the ribosome binding sites so that the genes could be placed under different promoters depending on desired output in different species.
The IAM pathway:
The IAM pathway taken for this module is from Pseudomonas savastanoi. This strain of soil dwelling bacteria is a known plant pathogen that uses IAA to infect its target. However, there have been some recent studies that suggest that IAA secretion by bacteria can also lead to positive microb-plant relations [1]. Therefore, we must carefully analyze what IAA concentration would aid root growth rather than promote gall formation. To achieve this, we will be experimenting with different levels of synthetic auxin on Arabidopsis thaliana. We will also be modelling this module in order to obtain the adequate concentration of IAA excretion from the chassis.
The effect on plants
To observe how indole 3-acetic acid influences plants, we will be working with the plant model organism Arabidopsis thaliana. Arabidopsis is well-established for research into plant biology and researchers have established lines that respond to auxin exposure by expressing reporter genes, which are particularly useful for our project.
We will use DR5:GFP and DR5:3XVENUS plants that respond to auxin by expression of GFP and YFP, respectively, to look at the plant response to synthetic auxin and later bacteria-secreted auxin. The DR5 plant lines respond to auxin exposure by expressing GFP and YFP, respectively. This will allow us to monitor how much auxin is taken up and which cells respond to it. We will be using confocal microscopy to evaluate the relative strength of fluorescence expressed by the plant. This will act as an indirect reporter on the auxin concentration supplied as it relies on the plant expressing fluorescence in response to stimulation by the hormone.
Initially, we will be supplying the plants with synthetic auxin and observe the differences in growth and (root) morphology due to differential concentrations of the hormone. In later stages of the project, this will be followed by exposing the plants to E. coli cells expressing auxin.
References: [1] Stijn Spaepen, Jos Vanderleyden, and Roseline Remans, “Indole-3-acetic acid in microbial and microorganism-plant signaling,” FEMS Microbiology Reviews 31, no. 4 (July 2007): 425-448.
