1.1 Salkowski assay

The Salkowski assay is a colourimetric assay that detects IAA with high specificity among other indoles. There are many different types of Salkowski reagents which work at different concentration ranges of IAA and with varying specificity. They all vary slightly in composition and measurement method. We used the most specific reagent according to a paper which works at a concentration range of 0 to 260µM. 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 DH5α (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 in the correct order of magnitude. E. coli are known to naturally express IAA, although the pathway is uncharacterised, which is why all of our controls showed moderate levels of IAA production^[1]. However, cells containing the Auxin Xpress construct have repeatedly shown to produce almost twice as much IAA.

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).

Due to the results of the light exposure test, all future cultures were incubated in the dark. In the assay comparing growth media (LB and tryptone broth) the OD of each sample, after 20 hours of incubation, was measured at 600 nm and appropriate dilutions of cell filtrate were made to normalise against the different cell densities of samples. The OD of cell filtrates after the addition of Salkowski reagent was measured at 530 nm and blanked with the appropriate growth medium for each sample. The OD measurements were then converted to IAA concentration with the standard Salkowski curve. Surprisingly, the results (Fig. 6) suggest that IAA production was optimal in LB, although the OD at 600 nm of cultures grown in tryptone broth (very nutrient rich) was much higher. We cannot draw a conclusion from this data, however it seems that the IAA producing pathway endogenous to E. coli is much more complicated than anticipated. We may postulate that IAA is not produced when growth conditions are very favourable and cell density is high.

The final Salkowski assay we tried was with E. coli BL21 transformed with the Auxin Xpress construct. This strain has two proteases knocked out and is favourable for protein over-expression. We left the liquid cultures to incubate in the dark for 20 hours and then measured OD as previously described. From this assay we got extremely high yields of IAA (shown by the bright red colour after addition of Salkowski reagent to cell filtrate, fig...) in the Auxin Xpress and control cells. We think that the assay was saturated with IAA and therefore tested the filtrate on HPLC to see if we could detect a difference in IAA production between the samples.

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 IAA'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. Imaging was carried out at precisely the same settings to ensure that the data could be compared and is representative of actual fluorescence.

Video 1. Response of the roots to IAA-expressing bacteria.

In order to quantify the expression of fluorescence in the plants exposed to different treatments we processed the images in ImageJ and analysed fluorescence intensity in individual particles. The relative fluorescence intensity of roots exposed to our auxin-secreting bacteria is 1.93x that of roots exposed to no bacteria and 2.9x that of roots exposed to non-auxin-expressing bacteria. It therefore appears that 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 IAA

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 2. 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 3. 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 4. 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.

From literature, the IAA concentration which promotes Arabidopsis root branching is centered within the range of 0.1 nM to 10 mM while the optimal concentrations are from 0.5 µM to 20 µM ^[2]. Accordingly, we supplied the roots with IAA concentrations ranging from 0.1 nM to 10 mM. The mentioned IAA concentrations were shown to inhibit root length as the concentrations decreases, therefore the concentrations were extended later down to 10 fM. Seedlings were grown for three days and imaged using a Fujifilm LAS3000 Imager. Data analysis was conducted using ImageJ (Fig. 1).

Plotting average root lengths (Figure 3) shows that IAA concentrations between 10 fM to 10¹¹ fM promoted root growth, whereas concentrations greater than 10⁹ fM stunted root growth. These results provide us with an approximate threshold IAA concentration of 10⁹ fM, past which Arabidopsis roots growth 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. The root branching could not be determined by the Imager since the plants were to small to observe. However, the confocal microscopy shows the root tip morphology at higher IAA concentrations (from 10⁹ fM onwards) to be much more fibrous than the control.

Arabidopsis seedlings against the different IAA concentrations in liquid media they were grown in show the similar pattern with the root length. This confirms that IAA could increase the plants' biomass and the increasing degree corelates well with the increase in root length.

Figure 9: Plot of biomass of 20 Arabidopsis's data collected demonstrating biomass of Arabidopsis as an approximate 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 growth. 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 10¹¹ fM. Accordingly, we added three different IAA concentrations ranging from 10⁸ fM to 10¹⁰ fM 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 10 : Phytogel plates applied with IAA concentrations of (a) 10¹⁰ fM (10 µM), (b) 10⁹ fM (1 µM), (c) 10⁸ fM (0.1 µM) and (d) control (0M) from left to right

Figure 11: 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 at distances further away. Accordingly, 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 and whether uptake into the roots would take place in nature. 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 with and without IAA supplementation, we set up a split root experiment as recommended to us by Dr Alexandru Milcu. This experiment extends beyond the scope of normal controls because the same plant is subjected to two different treatments in phytogel. We grew one half of the seedlings in phytogel containing three different concentrations of IAA and the other half in phytogel without IAA. Three replicates of seven day old Arabidopsis DR:3VENUS were set up for each concentration and grown for two weeks before measuring root length (Fig.1)

Figure 12: The split plates applied with IAA concentration of (a) 10¹⁰ fM (10 µM), (b) 10⁹ fM (1 µM), (c) 10⁸ fM (0.1 µM) and (d) and 10⁵ 0.1 nM

Figure 13: Curves showing the decrease of root length ratios by increasing IAA concentrations in split root experiment

Figure 14: 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 15: The split plates applied with IAA concentration of (a) 10⁴ fM, (b) 1,000 fM, (c) 100 fM pM, (d) and 10 fM The right half of the plates shows the control.

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 16: 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 10 fM, however, all of the concentrations lower than 10⁵ fM 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: 10¹¹ fM (0.1 mM), 10⁹ fM (1 µM), 10 ⁷ fM (10nM), 10⁵ fM (0.1 nM), 10 ⁴ fM, 1,000 fM, 100 fM, 10 fM (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.To measure how well the plant growth is, the number of germination were noted every 3 days as below.

As expected, the plants watered with IAA concentrations ranges of 10⁴ fM-10⁷ fM 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.

- 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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During the planning stage of our project we discussed several ways that the IAA could be delivered to the plant. In the end, we decided to either have the bacteria chemotax to the root and secrete the IAA around it or to have them be uptaken by the root through a natural process that has been recently documented in 2010. In order to determine whether the latter option is viable we have to look at the cell viability once it has been taken up into the root. In order to study that, we decided to use a photoconvertible protein called Dendra2.

Dendra2 is a photoconvertible protein that normally exhibits green fluorescence but can be irreversibly switched following irradiation at 405nm or 488nm to red fluorescence. This conversion cannot be reversed ^[4]. Dendra2 is therefore very useful for observing the metabolism of cells: if all protein in a cell is converted to red fluorescence and it subsequently expresses green fluorescence, the cell has to be metabolically active.

First we had to determine whether the excitation wavelength of the green fluorescense could photoconvert Dendra2. Since the excitation of the absorbance prior to photoconversion is 486nm and the photoconversion wavelength according to the literature is 488nm we had to make sure that a wavelength of 486nm would not cause photoconversion. In order to test for this, we excited cells with 486 nm and 558 nm wavelengths and measured the fluorescence at the emission wavelengths 505 nm and 575 nm.

The results show no detectable photoconversion meaning that the excitation wavelength of 488nm can be used. Once this was verified we attempted to look at the photoconvertability of Dendra2. We used a wavelength of 405nm and 505nm and then measured the emission at 575nm. Since the FluoroMax-3 machine we intended to use could not emit both of these wavelengths at the same time we had to seek the help of Tim Wilson who pried open the machine and modified it with his own 405nm laser pen and a filter from our gel imager. These modifications allowed us to obtain the following results.

We found that Dendra2 will fluoresce red upon conversion by light at a wavelength of 405 nm. Dendra2 reached its maximum conversion after only 200 seconds making it suitable for in vivo studies. We think that the degradation after the maximum conversion is due to photobleaching. We then decided to move on to something more interesting: testing whether Dendra2 will convert within a bacteria that has been taken up by a root cell!

Following the successful uptake of our Dendra2 expressing bacteria we conducted an in vivo photconversion study on the confocal microscope. Conversion with the 405 nm laser was completed after about 15 rounds of irradiation at 50% laser intensity with the pinhole set to 3 airy units.

As is visible from Video 1, protein in bacteria that are inside the area targeted by the laser switches from green to red fluorescence after about fifteen rounds of irradiation. One can also see a single bacteria that was left outside the targeted area that was not converted red. The switch from green to red fluorescence can be visualised much more acutely when observing the change in fluorescence in a single bacterium.

Due to time constraints, we were unfortunately not able to trace the metabolic activity of the cells inside the plant roots. However, we are planning to set up an experiment that consists of photoconverting Dendra2 in E. coli taken up by the roots and periodically image the same cells to assess whether they remain metabolically active once they are inside the plant. This is especially important as we would need to know whether the cells would be able to actively produce IAA inside the root cells.

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Since we have been working with the new photoconvertible fluorescent protein Dendra2 in order to do the in vivo imaging studies, we thought that it might be a good idea to characterize and compare other characteristics of this protein to the other fluorescent proteins in the registry. Since we were already working with sfGFP for our soil survivability experiments and root uptake experiments as well as mRFP1 for the Gene Guard we decided to use these as well as the standard GFPmut3b to compare their thermostability values. This study will give a good indication of which proteins will work best in thermophiles which will be useful for any future project involving thermophiles.

To characterise these parts, we determined the temperature at which these proteins denature and cease to fluoresce. The data was collected in two overlapping sets, ranging from 35°C to 66°C, and from 57°C to 96°C. This was because it was only possible to fit a maximum of eight samples into the thermocycler at once.

Once the data was collected by taking fluorescence readings from a 96-well plate, it was normalised to a sample of untreated cell lysate containing the fluorescent protein. This gave a value for Relative Fluorescence, which was plotted against temperature to create a scatter plot. A line of best fit was applied, and the mid point of the sigmoid region of the line was taken as the denaturation point of the protein.

The sigmoidal curves that were calculated gave us the following function in order to find K which we also call PTm50 (temperature at which half of the protein is denatured measured by looking at its fluorescence):

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[1]Ball, E.(1938)Hetroauxin and the growth of Escherichia coli. Journal of Bacteriology 36(5). pp. 559-565

[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.

[4] Gurskaya, N. et al. (2006) Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light. Nature Biotechnology 24, pp. 461-465.

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