Team:Imperial College London/Project Auxin Testing
Module 2: Auxin Xpress
Auxin, or Indole 3-acetic acid (IAA), is a plant growth hormone which is produced by several soil bacteria. We have taken the genes encoding the IAA-producing pathway from Pseudomonas savastanoi and expressed them in Escherichia coli. Following chemotaxis towards the roots and uptake by the Phyto Route module, IAA expression will promote root growth with the aim of improving soil stability.
How much IAA will we be producing? More importantly, has the module actually worked? We have used qualitative methods such as the Salkowski assay and quantitative methods such as HPLC and LCMS to determine the level of IAA produced by our construct. We have also done experiments on Arabidopsis root growth with synthetic IAA.
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 260 µM. 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 (Figure 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. (Data by Imperial College London iGEM team 2011).
Figure 2: Cuvettes used to measure OD for the standard curve. As IAA concentration increases, the solution progresses towards red. (Picture by Imperial College London iGEM team 2011).
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 (Figure 3&4), the results of which were positive for our IAA construct. The IAA producing E. coli were producing approximately 55 µM of IAA extracellularly. From modelling, we have determined that our construct would be able to produce 72.25 µM IAA intracellularly. E. coli are known to naturally produce IAA, which is why all of our controls show moderate levels of IAA production. However, cells containing the Auxin Xpress construct have repeatedly shown to produce almost twice as much IAA as control cells without.
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. (Data by Imperial College London iGEM team 2011).
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. (Data by Imperial College London iGEM team 2011).
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, tryptophan concentration and light exposure to optimize IAA production.
Testing the assay on synthetic IAA in LB after incubation overnigh in the dark compared to in the light confirms that IAA does degrade in the presence of light (Figure 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. (Picture by Imperial College London iGEM team 2011).
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. (Data by Imperial College London iGEM team 2011).
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 mixed with Salkowski reagent for each sample. The OD measurements were then converted to IAA concentration with the standard Salkowski curve. Surprisingly, the results (Figure 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, Figure 7) 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.
Figure 7: Salkowski assay of filtered supernatant from IAA expressing E. coli BL21 and control competent E. coli BL21 that have not been transformed. The extremely high levels of IAA produced seem to have saturated the reagent and therefore a conclusion cannot be drawn.
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Since the BL21 strain of E. coli was saturating the Salkowski reagent we looked for alternative quantification methods to measure the amount of IAA being produced by BL21 cells containing the Auxin Xpress construct. The extraction method we used resulted in a large loss in IAA from the cell filtrate and no peaks were detected by HPLC, therefore we ran filtered cell supernatant directly through the column and the peak characteristic to IAA at 227 nm was visible.
From the positive control, only one peak at 227 nm showed up at about 15 mins so we knew that this corresponded to IAA and looked for the same peak in our samples between 15 and 16 minutes.
Considerably smaller IAA peaks were present in the sample of interest and negative control and a difference in magnitude between the two could not be determined (Figure 8). Therefore we needed an even more sensitive approach to accurately determine how much IAA our engineered bacteria were producing.
Figure 8: HPLC peak corresponding to IAA. Positive control is 50 μM IAA in acetonitrile. Auxin Xpress is filtered supernatant of E. coli BL21 transformed with the Auxin Xpress construct. Negative control is filtered supernatant of E. coli BL21 transformed with a vector containing only a promoter and antibiotic resistance..
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Finally we did liquid chromatography mass spectrometry (LCMS) to confirm that IAA was present in our sample. This highly quantitative method combines HPLC and mass spectrometry allowing detection of nano-molar concentrations. Dr Colin Turnbull and his team kindly ran our samples for us. E. coli strain BL21 were used to maximise IAA output.
The characteristic IAA peak detected by LCMS confirms that IAA is being produced by cells with the Auxin Xpress construct (Figure 9). Unfortunately the extraction method resulted in a high loss of IAA in the sample (post extraction concentrations were nearly 12 fold lower than pre-extraction). Although the relative levels of IAA are still informative, we hope to optimise the extraction in the future to improve the yield of IAA.
Figure 9: Peaks produced from LCMS, the peak area of the MRM transition 176-130 was used to quantify IAA. The bottom window shows the peak for authentic IAA run in the same batch compared to the test sample extracted from our engineered bacteria. The peak around four and a half minutes corresponds to another unkown metabolite. The peak at about six and a half minutes corresponds to IAA.
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 the effects of indole 3-acetic acid (IAA). This will help us with the human practices aspect of our project, as it is part of product safety testing, 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.
Video 1. Response of the roots to IAA-expressing bacteria. (Imaging by Imperial College London iGEM team 2011).
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 0.1 mM IAA (imaging by Imperial College London iGEM team 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 Imperial College London iGEM team 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 0.01 nM IAA .(Imaging by Imperial College London iGEM team 2011).
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 10: 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 London 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. 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 (Figure 12).
Figure 11: 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 (10 nM), 105 fM (0.1 nM), 10 4 fM, 1,000 fM, 100 fM, 10 fM and control (no IAA) from left to right. (Data by Imperial College London iGEM team 2011).
Plotting average root lengths (Figure 13) 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. 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 12: Plot of root length data collected demonstrating root length of Arabidopsis as a bell-shape curve with increasing IAA concentrations in liquid medium. (Data by Imperial College London iGEM team 2011).
The biomass of the plants were also determined using the dry weight (Figure 13). The plot of the biomass of 20 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 13: Plot of biomass of 20 Arabidopsis plants. Results show biomass of Arabidopsis as an approximate bell shape curve with increasing IAA concentration in liquid media. (Data by Imperial College London iGEM team 2011).
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.(IAA was applied on the left side) 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 14: 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. (Data by Imperial College London iGEM team 2011).
Figure 15: Plot of root length data showing the effect of IAA concentration on root growth when applied at different distances from the seed. (Data by Imperial College London iGEM team 2011).
These results (Figure 15) 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.
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 (Figure 17)
Figure 16: 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. (Data by Imperial College London iGEM team 2011).
Figure 17: Curves showing the decrease of root length ratios by increasing IAA concentrations in split root experiment. (Data by Imperial College London iGEM team 2011).
In figure 17, 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 then carried out the experiment with lower IAA concentrations to observe the positive effect of IAA on root length. (Data by Imperial College London iGEM team 2011).
Figure 18: 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. (Data by Imperial College London iGEM team 2011).
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 19: Curves showing the decrease of root length ratios by increasing IAA concentrations in split root experiment. (Data by Imperial College London iGEM team 2011).
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.
2.7 Data fitting to support the IAA uptake and root growth modelling
A root growth rate of 0.96 cm/day with the external IAA concentration 5x10-5 mol/L is selected from the literature in order to simulate and visualise the root system (Click here to see the details of the root growth simulation). To get an accurate growth rate which is particularly fitting to our project, we decided to do data fitting analysis to the Arabidopsis we plant. The root length and number of branches of the Arabidopsis were recorded every two days from day 0 to day 9. Root length, daily root growth rate and number of branches are plotted against time and IAA concentration. Then the plots are analysed to give an approximation of the relationship between IAA concentration and root growth. The following graph gives an example of root length against time.
Figure 20: Root growth speed decays over time. This graph gives a prediction of the root growth speed (cm/day) for 20 days. The exponential decay constant is 0.048. (Data by Imperial College London iGEM team 2011).
Figure 21: Primary root length (mm) vs. time (day) and external IAA concentration (mol/L). The data fitting result gives this plot of the Arabidopsis primary root length. The length increases as the growth time increases, and reaches a maximal depth when the IAA concentration is approximately 1 pM. (Data by Imperial College London iGEM team 2011).
Figure 22: Primary root growth rate (mm/day) vs. root growth time (day) We used the data fitting toolbox of Matlab to obtain this figure. The relationship between the growth rate and the IAA concentration can be approximated by a Gaussian equation. The abnormality of the 0.1 nM curve is due to two contaminated samples which stopped growing at 7 mm after day 5. (Data by Imperial College London iGEM team 2011).
Figure 23: Number of lateral branches vs. external IAA (log) concentration. The optimal concentration for lateral root branching = 1 uM-10 nM, at this concentration, the Arabidopsis root gained the most lateral branches. (Data by Imperial College London iGEM team 2011).
The result of the data fitting does not coincide with our latest wet lab experimental results, since the optimal external concentration for Arabidopsis is found to be 0.1 nM. The failure of the previous experiment is analysed and gives the guidance for the new experiments in the following ways:
1. Instead of measuring the primary root length and number of lateral branches of Arabidopsis, the growth rate should be tested by measuring the dry mass of the roots.
2. At the early stage of planting, the number of lateral branches is very small, therefore massive percentage errors and standard derivation are produced.
3. Huge error is also introduced by the dilution process of the IAA solution to reach a low concentration level of 10-13 mol/L.
4. The wet lab result shows that the IAA concentration in phytogel media is negligible after 9 days, the degradation of IAA is an important factor when the root growth is modelled.
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 A. thaliana to retain the most soil. We exposed plants to different IAA concentrations ranging from 0.1 mM (1011 fM) to 10 fM. Fifteen 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. This compost was chosen to minimise errors resulting from different soil mixtures.
Figure 24: Experimental set up (A) 3 pots of Arabidopsis watered with 3 different IAA concentrations : 0.1 pM (left), 10 nM (middle), and 1 mM (right) (B) Set up of Arabidopsis growing in preparation for the erosion experiment.(Data by Imperial College London iGEM team 2011).
The effect of IAA can be seen at a very early stage. We observed the germination of the seeds that had been exposed to different IAA concentrations (Figure 25 and 26).
Figure 25: Germination speed and effectivity is affected by the by external auxin concentrations. (Data by Imperial College London iGEM team 2011).
Figure 26: Graph showing the correlation of the plants' ability to grow more than two leaves related to the external IAA concentration. (Data by Imperial College London iGEM team 2011).
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 concentrations higher than 10 nM and lower than 10 pM. 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).
We allowed the plants to grow for four weeks and then looked at the effect of IAA on Arabidopsis. More specifically, we wanted to look at whether there would be a difference in water retention within the soil. The water mass was obtained by measuring the difference between the ratio of the wet soil mass and the dry soil mass right after the Arabidopsis was watered and 3 days after being watered. The results are shown below .
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Figure 27: Water retainment by the soil with plants watered in different IAA concentration, control is soil without any Arabidopsis. (Data by Imperial College London iGEM team 2011).
These results correlate nicely with the bell-shaped curve obtained from the experiments on Arabidopsis in liquid culture. The optimal IAA concentration which gives the best water retetntion is between 0.1 nM - 1 nM.
While Arabidopsis is considered a fast growing plant, it takes several weeks to grow large enough 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. The plants took a total of five weeks to reach a size suitable for conducting the final erosion test. We set up the experiment to imitate water erosion as shown in the picture below.
Figure 28: Experimental set up of the erosion experiment. (Picture by Imperial College London iGEM team 2011).
Video 5: This video shows how the soil erosion experiment was set up and carried out.
The pots were cut open at one end to allow water with eroded soil to flow into the collecting basin. The slope was constructed by placing the pots on top of a 20 cm x 10 cm slanted platform to create a 30 degree slope. We applied water with a hose to imitate heavy rain fall. The pressure of water was kept constant by an adaptable shower head which imitated the average speed of rainfall (9 m/s) as suggested by Dr. Martin Selby. The shower head was placed about 90 degree to the ground and approximately 30 cm above the plant pot and water was applied for 10 seconds.
The eroded soil was collected from the basin and heated to evaporate the water so that we could measure the dry mass. The amount of dry mass collected gives us an indication of the amount of soil that would be swept away by water. Our results therefore show that soil erosion can be prevented through the use of vegetation that has been supplemented with IAA.
Figure 29: Mass of Soil eroded in different IAA concentration, control is soil supplemented with IAA but without Arabidopsis. The small box is a zoomed in image of the same graph without the control and 0M IAA applied and shows visually the bell shape that correlates to previous data on root length with varying IAA concentration. (Data by Imperial College London iGEM team 2011).
The results show a similar pattern with the water retainment results. In both of these results, 0.1 nM appears to be the optimum. At this concentration, the mass of soil eroded by water was the least and the water retention was the highest. Compared to the control with Arabidopsis and no IAA supplementation, the optimal IAA could improve soil retention by a factor of 100. Moreover, the optimal IAA concentration could decrease the mass of the soil eroded by a factor of 200 when compared to a control where soil in the absence of Arabidopsis was irrigated with IAA supplemented water.
After the experiment, the plants were collected from each IAA concentration and incubated to obtain the dry mass. The results are shown below.
Figure 30: Biomass of plants grown in different IAA concentrations (Data by Imperial College London iGEM team 2011).
The results of the biomass show a similar trend with the water loss, mass of the soil eroded and the biomass in liquid media. This allows us to support the viability of our project. This proves that IAA does increase plant biomass at optimal concentrations. These results have a huge implies that our system could be used in other applications such as improving crop yields.