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^[5]. 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 cells.

Due to inconsistent results with colour change on various occasions, we redid the Salkowski assay with repeats where we varied certain conditions. 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.

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 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 and therefore we will need to test the supernatant with HPLC.

1.2 HPLC

Collapse

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 IAA 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.

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.

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

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

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 producing to optimise root growth.

Figure 7: Arabidopsis grown in liquid media (A) 3 flasks of Arabidopsis in liquid media containing 3 different IAA concentrations : control (left), 0.1 pM (middle) and, 0.1 nM (right) (B) Set up of Arabidopsis grown in liquid media. (Photos by Imperial College iGEM team 2011).

The literature states that Arabidopsis is sensitive to IAA concentrations of 0.1 nM - 10 mM with increased branching seen between 0.5 µM - 20 µM ^[2]. In our experiments, we initially tested the effect of IAA concentrations of 0.1 nM - 10 mM. Unfortunately, our negative control (seedling with no additional auxin) got infected by a fungus preventing us from determining whether IAA could enhance root growth. Analysis of the uninfected seedlings revealed that increasing IAA resulted in decreased root growth. In response to this finding, we repeated the experiment with an extended IAA range of 0.01 pm - 10 mM. Within this extended range, we were able to find the optimum concentration of IAA for enhanced root growth. Seedlings were grown for three days and imaged using a Fujifilm LAS3000 Imager. Data analysis was conducted using ImageJ (Fig. 1).

Figure 8: The effect of IAA on root length. (A1) shows root growth of plants supplied with 10¹¹ fM IAA and (A2) with 10⁹ fM IAA (B1) shows the root growth with 10⁷ fM IAA and (B2) with 10⁵ fM IAA. (C1) shows root growth of plants supplied with 10 ⁴ IAA and (C2) with 1,000 fM IAA (D1) shows root growth of plants supplied with 100 fM IAA and (D2)with 10 fM IAA (E) Comparing the effect of root growth with 10¹¹ fM (0.1 mM), 10⁹ fM (1 µM), 10⁷ fM (10nM), 10⁵ fM (0.1 nM), 10 ⁴ fM, 1,000 fM, 100 fM, 10 fM and control (no IAA) from left to right.

Plotting average root lengths (Figure 3) shows that IAA concentrations between 10 fM to 0.1 mM 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 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.

Figure 9: Plot of root length data collected demonstrating root length of Arabidopsis as a bell shape curve with increasing IAA concentration in liquid media.

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.

Collapse

3.1 Soil erosion experiment

As promoting root growth to anchor top soil is the main objective of our project, we set up a soil erosion experiment to evaluate the effect of IAA on Arabidopsis thaliana's ability to hold down soil. In particular, we wanted to know which concentration of IAA causes the optimal vertical and lateral root growth. We exposed plants to a gradient of IAA concentrations ranging from 0.1 mM (10¹¹ fM) to 10 fM. 15 plants were seeded per pot and two replicates were set up for each concentration. We chose to plant our seeds in M2 compost which consists of organic compost and sand but does not contain pebbles or gravel. This compost was chosen to minimise errors resulting from different soil mixtures. M2 imitates the normal soil found in drylands.

Figure 17. Experimental set up (A) 3 pots of Arabidopsis watered with 3 different IAA concentrations : 0.1 pM (left), 0.1 nM (middle), and 1 mM (right) (B) Set up of Arabidopsis growing in preparation for the erosion experiment.

The effect of auxin can be seen from very early stages onwards: we observed germination of seeds that had been exposed to different IAA concentrations (Fig. 17) as an indication of plant growth speed and effectivity (Fig. 18).

Figure 18: Germination speed and effectivity depend on the auxin concentration supplied.

Figure 19: Dependence of the plants' ability to grow more than two leaves on IAA concentration.

Plants watered with IAA concentrations ranging from 10 pM to 10 nM germinated first and produced a higher number of leaves than plants exposed to higher or lower concentrations of the compound. The growth trend caused by differing IAA concentrations in soil is quite similar to that observed in liquid media. However, the ideal IAA concentration for plant growth is higher in soil than in liquid media (1 nm rather than 0.1 nm). This might result from higher dispersal and degradation of IAA in soil.

After the plants were grown for 4 weeks, the soil was collected to determine the water mass retained in the soil which correlates to the root mass.

While Arabidopsis is considered a fast growing plant, it takes several weeks to grow sufficiently big to hold down soil effectively. This effect is exacerbated by the fact that we are growing several plants in one pot, thereby increasing the competition for nutrients. Our plants are still too young for observing their soil conservation capabilities. We are expecting the plants to take a total of four to five weeks to reach a size suitable for conducting the erosion experiment. We will need to amend the experimental setup to imitate water erosion: the pots will be tilted to imitate a slope and water will be applied at a set and constant pressure to imitate rain fall (Figure 20).

Figure 20: Experimental set up of the erosion experiment.

In further development of the project, the pots will be cut open at one end to allow water with eluded soil to flow into the collecting basin. Slopes will be constructed by placing the pots on top of 20 cm x 10 cm baskets to create a 30 degree slope. We will apply water to imitate rain fall that strong enough to sweep away the soil. The pressure of water will be kept constant by an adaptable shower head which imitates the average speed of rainfall (9.8 m/s). The shower head is placed 90 degrees to the ground and 30 cm above the top of the plant pot.

The eroded soil will be collected from the basin and incubated to measure the dry mass. The amount of dry mass collected will give us an indication of the amount of soil swept away by the water and therefore if supplementation of IAA to the soil decreases the amount of soil eluded.

We are hoping that we will be able to collect data on this experiment prior to the regional Jamboree in Amsterdam.

Collapse

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.

Fig. 3:

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.

Fig. 3:

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.

Figure 4. Dendra2 photoconversion in bacteria taken up inside plant roots. The graph on the top displays averaged fluorescence over the entire photoconverted area and the amount of brightfield light (the background light used to see the outline of the roots) recorded is therefore very high. The graph on the bottom displays emission at green and red fluorescence over the same time span.

Video 1. This video shows the photoconversion of Dendra2 within E. coli cells that have been taken up into the plant roots as a time-lapse of pictures taken after each round of irradiation at 405nm. The targeted area of cells being photoconverted corresponds to the top graph in Figure x. There is a single bacterium visible on the right that was not targeted for photoconversion and serves as a control (data and imaging by Imperial College iGEM 2011).

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.

Collapse

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.

Fig. 4: Results of the heat denaturation experiment. The temperature at which half of the protein is denatured measured by looking at its fluorescence (PTm50) mRFP1: 82.2°C; GFPmut3b: 61.6°C; Dendra2: 89.1°C; sfGFP: 75.0°C.

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):

Collapse

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

[1] Spaepen S, Vanderleyden Jos and Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiology Reviews 31(4): 425-448.

[2] King JJ, Stimart DP, Fisher RH and Bleecker AB, (1995) A mutation altering auxin homeostasis and plant morphology in Arabidopsis. The plant cell 7: 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: 461-465.

[5] Gordon, S. & Weber, R. (1951) Colorimetric estimation of indoleacetic acid. Plant Physiology 26: 192-195.

Collapse