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).

Figure 5: Testing the effect of light exposure on synthetic IAA stability. The cuvette on the left shows the colour change at point zero. The three middle cuvettes were left in the dark overnight and the three on the right were left exposed to light, after which Salkowski reagent was added to all samples to observe colour change.

Figure 6: Salkowski assay performed on IAA producing E. coli and control E. coli incubated for 20 hours in different media and at two different temperatures. All samples were incubated in the dark.

From the results of the light exposure test, all future cultures were incubated in the dark. In the assay comparing growth media and incubatin temperature, 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 IAA on plant roots, we applied different concentrations of synthetic IAA to Arabidopsis seedlings in liquid culture. The results of this experiment have given us an idea of the ideal concentration of IAA that our engineered bacteria should be expressing to optimise root growth.

Figure 1: Arabidopsis grown in liquid media

From literature we know that the IAA concentration optimal for Arabidopsis root growth is within the range of 0.5 µM to 20 µM but has an effect at the wider range of 0.1 nMto 10 mM ^[2]. Accordingly, we supplied the roots with IAA concentrations ranging from 0.1 nM to 10 mM to provide a larger data range for analysis. Seedlings were grown for three days and imaged using a Fujifilm LAS3000 Imager. Data analysis was conducted using ImageJ (Fig. 1).

Figure 2: The effect of IAA on root length. (a) The top panel shows root growth of plants supplied with 0.1 mM IAA and the bottom panel with 1 µM IAA (b) The top panel shows the root growth with 10 nM IAA and the bottom panel with 0.1 nM IAA. (c) The top panel shows root growth of plants supplied with 10 pM IAA and the bottom panel with 10 pM IAA (d) The top panel shows root growth of plants supplied with 0.1 pM IAA and the bottom panel with 0.01 pM IAA (e) Comparing the effect of root growth with 100,000 nM (0.1 mM), 1000 nM (1 µM), 10 nM, 0.1 nM, 10 pM, 1 pM, 0.1 pM, 0.01 pM and control 0 pM IAA from left to right.

Plotting average root lengths (Figure 3) shows that IAA concentrations between 0.01 pM to 10 nM promoted root growth, whereas concentrations greater than 10 nM, stunted root growth. These results provide us with an approximate threshold IAA concentration of 10 nM, past which Arabidopsis roots will be negatively affected (Figure 2). We can use this value to optimise IAA production by our Auxin Xpress construct by switching the promoter and RBS without surpassing the threshold value.

Figure 3: Plot of root length data collected demonstrating root length of Arabidopsis as a bell shape curve with increasing IAA concentration in liquid media.

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

The effect of IAA on root growth was also tested in phytogel by dotting it at varied distances from the root to simulate bacteria producing IAA. 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 from around the root in the soil without being taken up by the root. We used DR:3VENUS seeds. These germinate into plants whose roots respond to IAA by expressing YFP.

From modelling we estimated the level of IAA secretion from our bacteria to be 0.01 mM. Accordingly, we added three different IAA concentrations ranging from 0.1 µM to 10 µM IAA at five distances ranging from 2 to 10 cm from the seed. Five replicates were set up for each concentration. The roots grew perpendicular to the line on which IAA and Arabidopsis seeds were applied. The phytogel plates were kept in the dark and at 4°C for three days in order to prevent the degradation of IAA. They were then transferred into light for another six days to observe root growth. After nine days total, the phytogel plates were imaged and root lengths measured.

Figure 1 : Phytogel plates applied with IAA concentrations of (a) 10 µM, (b) 1 µM, (c) 0.1 µM and (d) control (0M) from left to right

Figure 2: Plot of root length data showing the effect of IAA concentration on root growth when applied at different distances from the seed.

These results (fig 2) suggest that IAA can have a promoting effect on root growth in close proximity to the seed, as well as distances further away. Therefore whether our system promotes or inhibits root growth is influenced both by the position of the bacteria relative to the root and the amount of IAA produced by the bacterial population. Future optimisation of IAA production levels therefore depends on the affectivity of the Phyto-Route module and how close to the root our bacteria would swim in the soil. In future iterations of the design cycle, this data could influence both Phyto-Route and Auxin Xpress.

2.6 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.

Figure 1 : The split plates applied with IAA concentration of (a) 10 µM, (b) 1 µM, (c) 0.1 µM, (d) and 0.1 nM

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

Curves showing the decrease of root length ratios by increasing IAA concentrations in split root experiment

Figure 2 : 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.

Figure 3 : The split plates applied with IAA concentration of (a) 10 pM, (b) 1 pM, (c) 0.1 pM, (d) and 0.1 pM

The ratios between the IAA samples and the controls at each concentration were calculated and plotted into a graph as the same as mentioned above

Figure 4 : Curves showing the decrease of root length ratios by increasing IAA concentrations in split root experiment

Even though in phytogel, the root length decreasing trend is shown quite clearly and reach the maximum at 0.01 pM, however, all of the concentrations lower than 0.1 nM obviously shows higher root length than the control.

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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.

Figure 1 : 15 of Arabidopsis are grown in each of the 30 pots, where 3 of each are watered with the same IAA concentration.

The Arabidopsis seeding in the soil was carried out as mentioned in the protocol.To measure how well the plant growth is, the number of germination were noted every 3 days as below.

Figure 2 : The graph showing the number of the germinating plants watered with different IAA concentrations

Figure 3 : The graph showing the number of the germinating plants with more than 2 leaves watered with different IAA concentrations

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. The trend of the growth with different IAA concentrations in soil is considered quite similar to the liquid media.

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.

Figure 4 : The diagram representing the setup of soil erosion experiment

- 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 as mentioned in the protocol. The higher the dry mass collected, the more soil is eroded.

However, since Arabidopsis take quite a long time (at least 4-5 weeks) to grow enough for soil holding up. The fact that many plants were grown in a pot makes them compete with each other for the nutrient and grow more slowly than in individual. The results are therefore expected to be completed by 1-2 weeks after the wiki freeze which might be shown during the regional jamboree.

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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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