1.1 Salkowski assay

The Salkowski assay is a colourimetric assay that detects IAA with high specificity among other indoles. There are three different types of Salkowski reagents which work at different concentration ranges of IAA and with varying specificity. We used the most specific reagent which works at a concentration range of 0 to 45 µg/ml. Modelling of the Auxin Xpress construct informed us that IAA production would be within this range. This standard assay is the simplest way to determine whether there is IAA present in solution. First we created a standard curve with increasing IAA concentration in LB broth using synthetic IAA (Fig. 1&2). This was used to determine IAA concentration from OD measurements of IAA-producing E. coli.

The aim of this experiment was to determine whether our construct is working and roughly how much IAA we are producing when compared to control cells without the construct.

Figure 1: Standard curve of Salkowski assay made with synthetic IAA in LB

Figure 2: Cuvettes used to measure OD for the standard curve. As IAA concentration increases, the solution progresses towards red.

We did preliminary tests with our IAA producing cultures with a spectrophotometer. Once IAA presence was confirmed by colour change, we set up a more thorough assay with a BMG Omega plate reader. The first assay we did was with E. coli DH5 (Figs. 3&4), the results of which were positive for our IAA construct. The IAA producing E. coli were producing approximately 55 uM of IAA. From modelling, we have determined that our construct would be able to produce 72.25 uM IAA, which shows that we were are in the correct order of magnitude.

Figure 3: Results from trial 1 of Salkowski assay with cell filtrate of IAA producing E. coli DH5α. Filtered through a 0.2 µm pore filter

Figure 4: Visual results correlating with OD measurements. The eppendorf on the right contains IAA producing E. coli DH5α and the eppendorf on the left contains control E. coli DH5α.

Due to inconsistent results with colour change on various repeats, we redid the Salkowski assay with repeats using different controls. We tested different growth media, incubation temperature, tryptophan concentration and light exposure to optimize IAA production.

Interestingly, from testing the Salkowski assay on synthetic IAA in LB left overnight in dark versus light suggests that light exposure does lead to IAA degradation (Fig. 5).

The OD of each sample was measured at 600 nm and appropriate dilutions of cell filtrate were made to normalise against different growth rates. OD of cell filtrates with Salkowski reagent were measured at 530 nm and blanked with the appropriate growth medium for each sample. The OD measurements were then converted to concentration with the standard Salkowski curve (fig. 6). Surprisingly, the results suggest that IAA production was optimal in LB, although the OD at 600 nm of cultures grown in tryptone broth (very nutrient rich) were much higher.

1.2 HPLC

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2.1 Confocal imaging with fluorescent reporter cell lines

We are not only interested in constructing the IAA-producing pathway in our bacteria but we also want to investigate what effect the IAA has on plants to verify our assumptions about indole 3-acetic acid (IAA)'s effects. This will help us with the human practices aspect of our project and it will also provide a good assay for the functionality of IAA-secreting bacteria.

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 IAA exposure by expressing reporter genes, which are particularly useful for our project.

We used DR5:3XVENUS plants that respond to IAA by expression of YFP to look at the plant response to synthetic IAA and bacteria-secreted IAA. This will allow us to monitor how much IAA is taken up and which cells respond to it. We will be using confocal microscopy to image and evaluate the relative strength of fluorescence expressed by the plant. This type of imaging is especially useful as it makes three-dimensional imaging of samples possible thanks to precise imaging of individual points. The fluorescence acted as an indirect reporter on the IAA concentration supplied as it relies on the plant expressing fluorescence in response to stimulation by the hormone.

2.2 Effect of IAA produced by our bacteria

Once we had engineered our bacteria to express IAA, we an experiment to investigate the auxin's effect on the roots. This was done by supplying the plants with bacteria that produce IAA. Plants that were not exposed to bacteria showed strong autofluorescence in response to the IAA naturally produced in the roots. However, the roots supplied with bacteria unable to express IAA only exhibited very weak fluorescence. Plants supplied with IAA-expressing bacteria were brighter than either of the two controls.

Video 4. Response of the roots to IAA-expressing bacteria.

It therefore seems as though bacteria normally suppress production of IAA in root tips. However, the bacteria we have engineered to express IAA produce enough of the compound to not only overcome this limitation but to also increase expression of the reporter gene.

2.3 Effect of synthetic auxin

We exposed Arabidopsis seedlings to different concentrations of synthetic IAA.

Plants incubated with 0.1mM of IAA showed strongly enhanced lateral root growth but also stunted growth. This does not come as a surprise as concentrations this high are usually considered detrimental for the plant.

Video 1. Z-stack imaging of A. thaliana roots exposed to 0.1mM IAA (imaging by ICL iGEM 2011)

At lower concentrations, stunted growth was not observed. However, root tip cells were very brightly fluorescent and increased root branching could be observed.

Video 2. Root of A. thaliana seedling incubated with 1uM indole 3-acetic acid (imaging by ICL iGEM 2011).

At even lower concentrations, fluorescence was almost impossible to detect and much less branching of the root can be observed.

Video 3. Response of the roots to 0.01nM IAA (Imaging by ICL iGEM 2011).

2.4 Effect of IAA concentrations on Arabidopsis root growth

To assess the effect of auxin on plants, we applied different synthetic IAA to Arabidopsis seedlings in liquid culture. The response of the plants to different concentrations of the compound gave us an idea of the ideal concentration of auxin our bacteria should be expressing for optimal root growth.

We modelled the concentration of IAA secreted by our bacteria to be 72.25 µM. This is within the same order of magnitude as the ideal IAA concentration for the promotion of root growth found in literature (0.5 µM to 20 µM) ^[2]. Accordingly, we supplied the roots with IAA concentrations ranging from 0.1 nM to 10 mM. Plants were grown for three days and subsequently imaged using a Fujifilm LAS3000 Imager. Data analysis was conducted using ImageJ (Fig. 1).

The average root length was plotted to illustrate the inhibiting effect of high IAA concentrations on root growth (Fig. 2). This will allow us to determine what concentration of IAA should be produced by our bacteria and therefore allow us to choose the correct promoter.

The experiment is extended further down to 0.01 pM IAA concentration to investigate the promotion of root growth.

2.3 The effect of IAA distance from the root tip on root growth.

The effect of IAA on root growth was also tested in phytogel. On these gels, individual seedlings grow horizontally into a gel containing plant nutrients. These gels enable us to supply the plant with IAA at set distances from the seedling itself. From these experiments we hoped to determine whether our IAA producing bacteria will have an effect on plant root growth without being taken up by the root cells. We used DR:3VENUS seeds. These germinate into plants whose roots respond to IAA by expressing YFP. We attempted to get an estimate of the effect of IAA by comparing intensities of fluorescence across plants supplied with different concentrations of IAA.

Our modellers corrected the estimated IAA secretion of our bacteria from 0.0001 to 0.01 mM. Accordingly, we set up the IAA concentration gradient experiment with 0.0001 (0.1 µM), 0.001 (1 µM) and 0.01 mM (10 µM)of IAA at distances ranging from 2 to 10 cm from the seed. Five replicates were set up for each concentration. Roots grew perpendicular to the line on which IAA and seeds were applied. The plates were kept in the absence of light for 3 days in order to prevent the degradation of IAA and also in 4°C. The plates were later put in light for another 6 days to see root growth.

After 6 days, the phytogel plates were imaged with a Fujifilm LAS3000 Imager. The root length data collected was analyzed using ImageJ. The data was plotted to see the root length for each IAA concentration.

10 µM, 1 µM, 0.1 µM and control (0 M) of IAA was applied at 2 cm, 4 cm, 6 cm, 8 cm, 10 cm away from the seeds from left to right.

The average root length for each of the different seed to IAA source distances and IAA concentrations were plotted to study the effect of different IAA concentrations and distances of application on root length. The information could help us to predict the optimum promoter strength for the system.

Here, the results show the optimal distances away from seedlings in the range of 2-10 cm listing at below 0.1 µM : 2 cm, 1 µM : 6-8 cm, 10 µM : 6-10 cm. The results support the root length promoting effect by 0.1 µM of IAA since the root grows more in the nearer proximity to the IAA applying position. Also the results suggest the inhibiting effect from IAA concentrations of 1 µM and 10 µM (higher extent in the latter) since root length decreases in shorter distance from applying position but increases as the concentrations are diluted by longer distances.

2.4 The split root experiment

To visually compare the difference of the root growth between applying and non-applying of IAA in the same plant, we set up a split root experiment (which had been recommended to us by Dr Alex Milcu). This experiment extends beyond the scope of normal controls as the same plant is subjected to two different treatments. We supplied the following concentrations of IAA to one half of the roots: 0 µM (control), 0.1 µM, 1 µM and 10 µM, while the other half was grown in phytogel containing no IAA. 3 replicates were set up for each concentration. 7-day old seedling of A thaliana DR:3VENUS were used for this set up. This strain responds to IAA by expressing YFP. The plates are sealed and kept in the incubating room for 2 weeks in order to observe the length of the grown root.

After 2 weeks, the phytogel plates were imaged with a Fujifilm LAS3000 Imager. The maximal root length data collected were analyzed using image J program by comparing with the reference.

Below are remaining split root plates which were not infected by the fungus. The split plate contains the control half (on the right side of the plate) and a half where different concentrations of IAA are applied.

The length of the roots analysed using ImageJ are written on the table, which could be used to calculated the ratio between the IAA applied samples and the controls.

The ratios between the IAA samples and the controls at each concentration were calculated and plotted into a graph below

The ratio is shown to fit logarithmically with increasing IAA concentrations. However, the inhibiting effect found in phytogel is higher than the liquid medium. We were then carrying on the experiment with lower IAA concentrations to observe the positive effect of IAA in root length.

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3.1 Soil erosion experiment

As the main objective of the project is to promote root growth in order to prevent soil erosion and desertification by improving soil holding up. In order to determine the best root architecture (in terms of length and dispersion) for fixing the soil and retaining moisture inside the soil, we used 8 concentrations: 0.01 pM, 0.1 pM, 1 pM, 10 pM, 0.1 nM, 10nM, 0.001mM, and 0.1mM (same as the effect of different IAA concentrations experiment). These different IAA concentrations allow the roots to show different architectures.
The Arabidopsis seeding in the soil was carried out as mentioned in the protocol.The plant growth and the number of leaves were noted every 3 days as below.

The data on the number of plants grow with fewer or equal to 2 leaves (shown in black) and ones with more than 2 leaves (shown in red) watered with different IAA concentrations are tabulated below.

As expected, the plants watered with IAA concentrations ranges of 10 pM-10 nM emerge first from the ground and produce more number of leaves than the others.

After the plants were grown for 4 weeks. The soil is collected for obtaining mass of the water retained in the soil as said in the protocol. .

To see how rooting could prevent soil erosion. The experiments were carried out as shown in the diagrams below.

- the soil basket was cut opened at one end to allow water with eluded soil to flow into the basin. - The slopes were made by placing 20 cm x 10 cm baskets on top of the 30 degree slope.
- The soil chosen is called M2 which composes of organic compost and sand without any gravel to eliminate the error from different mixtures contributing to different soil contents. M2 imitates the normal soil found in degrading area.
- The pressure of water is kept constant by the adaptable shower head which imitates the average speed of rainfall (9.8 m/s). The shower honk is placed at 90 degree to the floor 30 cm above the top of the plant pot.

The eroded soil collected from the basin was incubated to measure the dry mass. The higher the dry mass collected, the more soil is eroded.

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[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.

[2] Joseph J. King, Dennis P. Stimart, Roxanne H. Fisher, and Anthony B. Bleecker, ''A mutation altering auxin homeostasis and plant morphology in Arabidopsis'', The plant cell, Vol.7(December 1995), 2023-2037.

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