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From 2011.igem.org

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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.

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Testing

How much auxin will we be producing? More importantly, has the module actually worked?! This chapter will look into the methods that we have decided to use in order to measure the amount of auxin in a solution. We have decided to use qualitative methods such as the Salkowski reagent (changes colour which is always good) as well as quantitative methods such as HPLC and GC-MS for more accurate results. Cross our fingers and hope this works!

26th of August

Colony PCR results of CPEC assembled PA2652 construct look promising! Will know for sure when sequencing results arrive next week

Gel 12: Colony PCR of 19 colonies picked from cells transformed with CPEC assembled PA2652 construct, about half have the correct size insert, these will be inoculated and miniprepped. Gel 13: Two more colony PCRs which were unsuccesfull, followed by five colony PCRs from negative control colonies (assembly of backbone vector without insert)showing backbone vector (6a) in one. The next well is a positive control colony PCR with plasmid 6a. The final two wells are analytical PCRs of the CPEC assembly and negative control with sequencing primers showing the correct size band for the assembled insert.

5th of August

After successfully testing the S2/1 method we also attempted the PC method which is both more exact and more specific for IAA. However, it only works at a lower range of concentrations of IAA and will therefore be useless if our bacteria end up excreting more than 20 μg/ml of IAA into the solution. Either way, we now have two standard curves which can be used to measure the amount of IAA in a solution. We are ready for the synthetic genes to arrive!

8th of August

Today we performed the first PC Salkowski assay successfully. We even took a picture of the gradient:

This was one of the 3 repeats that gave us the following standard curve:

26th of August

Today we have determined that the Salkowski reagent that we are currently using is not specific enough to detect indole-3 acetic acid. Instead, it is reacting with random indoles found within the supernatant and the cell samples. After working the entire day in order to obtain a conclusive answer on whether the cells are producing auxin or not has led to a frustrating result where the current reagent's only use is that of a very elaborate cell density measurement system, ergo useless. We will attempt HPLC next week as well as do some further reading on what we might have done wrong.

2nd of September

Today we attempted the Salkowski reagent that consists of iron (III) chloride and perchloric acid. This reagent seems to work and our cells seem to be producing auxin!

Implementation

Indole-3 acetic acid (IAA) is one of the most well studied phytohormones and is also known more commonly under the name Auxin. IAA is known as a key player in the regulation of plant growth and is also a known morphogen implicated in a vast array of processes ranging from embryo patterning to isodiametric expansion (fruit growth).

However, the topic of auxin producing soil bacteria in the rhizosphere has been given little attention so far. We believe that plant-microbe interactions mediated through IAA could be tapped into to modulate the plasticity of the root architecture. In this module, we will be attempting to express Tryptophan monooxygenase (IaaM) and Idoleacetimide hydrolase (IaaH) in Escherichia coli. We are aware that E. coli would not be a suitable chassis for field work and we have taken this into account when we made our DNA sequences.

The effect on plants

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

We will use DR5:GFP and DR5:3XVENUS plants that respond to auxin by expression of GFP and YFP, respectively, to look at the plant response to synthetic auxin and later bacteria-secreted auxin. The DR5 plant lines respond to auxin exposure by expressing GFP and YFP, respectively. This will allow us to monitor how much auxin is taken up and which cells respond to it. We will be using confocal microscopy to evaluate the relative strength of fluorescence expressed by the plant. This will act as an indirect reporter on the auxin concentration supplied as it relies on the plant expressing fluorescence in response to stimulation by the hormone.

Initially, we will be supplying the plants with synthetic auxin and observe the differences in growth and (root) morphology due to differential concentrations of the hormone. In later stages of the project, this will be followed by exposing the plants to E. coli cells expressing auxin.

References:
[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.

Chapter 3: effect of auxin on plants

We are not only interested in constructing the auxin-producing pathway in our bacteria but we also want to investigate what effect the auxin has on plants to verify our assumptions about indole 3-acetic acid'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 auxin-secreting bacteria.

Root growth at different auxin concentrations. Tuesday, 9 August 2011

To look at the effect of auxin on plants, we supplied differing indole 3-acetic acid concentrations to Arabidopsis seedlings in liquid culture.

Si modelled the concentration of auxin secreted by our bacteria to be 10mM. However, according to Joseph et al (1995), increasing exogenous IAA concentrations from 0 - 0.1 nM increases root growth from 0 - 20%. From 0.1 nM to 10microM, the root length and decreases sequentially, while fibrosity increases. The plant dies at concentrations over 10 microM. However ___ state that the optimal auxin concentration lies in between 0.5 microM - 20 microM. Accordingly, we used concentrations starting from 10mM to 0.1nM to test the effect of different auxin concentrations on the length of the roots and their branching. We made the auxin concentrations by serial dilution and added 10ml of concentrated auxin solution to 100ml of half-MS media each. Twenty-five seeds were added to each flask. The seeds were incubated at 23°C and wrapped in aluminium foil to allow the plants to germinate in the dark. They will be allowed to grow in the light in 3 days' time. This follows a protocol described by King et al. (1995). The results are stated in Friday 19th of August and Monday 22th of August

Root growth at different positions where Auxin is applied Wednesday, 10 August 2011

Looking at the effect of auxin on the roots also involves observing its effect on phytogels. On these gels, individual seedlings grow horizontally into a gel containing plant nutrients. These gels enable us to supply the plant with auxin at set distances from the seedling itself. Observing these effects is especially in case our bacteria do not get taken up into roots in nature, which has yet to be investigated. We are using DR:3VENUS seeds. These germinate into plants whose roots respond to auxin uptake by expressing YFP. We hope to be able to get an estimate of the effect of auxin by comparing intensities of fluorescence across plants supplied with different concentrations of auxin.

Si corrected the estimated auxin secretion of our bacteria to 0.0001 to 0.01 mM. Accordingly, we set up the auxin concentration gradient experiment with 0.0001, 0.001 and 0.01 mM of IAA. We injected a small volume of IAA dissolved in 70% ethanol at set distances from seeds, which were subsequently put onto the gel. Five replicates were set up for each concentration. On each plate, the seed seeds are sown at distances of 2,4,6,8, and 10 cm from the point where IAA is applied. Roots grow perpendicular to the line on which IAA and seeds are applied. The plates are kept in low light for 3 days in order to prevent photooxidation of auxin and also in 4 C to simulate the winter hibernation. The plates are later put in light for another 6 days to see root growth.

The split root experiment (Friday 12 and Monday 15 August 2011)

To visually compare the difference of the root growth between the 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 microM (control), 0.1 microM, 1 microM and 10 microM, while the other half is 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 auxin 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.

Friday, 19 August 2011

We imaged the plants that had been incubated with differing concentrations of IAA using confocal microscopy. Plants incubated with 0.1mM of IAA showed strongly enhanced lateral root growth but also stunted growth.

A Z stack through an Arabidopsis root tip incubated with 0.1mM indole 2-acetic acid.

This did not occur at lower concentrations. However, fluorescence was still clearly visible:

Root of A. thaliana seedling incubated with 1uM indole 3-acetic acid.

At even lower concentrations, fluorescence was much weaker:

Incubated at 0.01nM.

Effect of auxin concentrations results Monday, 22 August 2011

The results shows that from low (0.1nM) to high concentrations (0.1mM)the root legth and the number of the leaves decrease seqeuntially and the plants all die at 10mM. This is an important information for the modelling team since auxin only operates well at liquid concentrations less than 0.1nM. To find the optimal concentrations of auxin for maximal root growth and splitting. We extend the contrations from 0.1 nM lower down from 10 pM, 1 pM, 0.1 pM and 0.01 pM. Hopefully we could produce the bell shape curve graph for both the root's length and its fibrousity

Effect of auxin concentrations in phytogel Tuesday, 23 August 2011

In order to modelling the effect of auxin concentrations on Arabidopsis and build the macros on lateral root initiation and elongation, root length should be measured day by day. This could not be done in liquid media since taken the plant out of liquid media makes the plant prone to fungal contamination. Also the resources we have, e.g. number of shakers, amounts of Venus Arabidopsis seeds are not enough to build up repeats to measure root length everyday. Therefore we decide to grow Arabidopsis in phytogel which is less reliable than the liquid media

Soil erosion experiment Thursday, 25 August 2011

In order to see which root architecture, the length and the dispersion, is the best to hold up the soil to prevent soil erosion and retain the moisture inside the soil. We then vary 8 concentrations from 0.01 pM, 0.1 pM, 1 pM, 10 pM, 0.1 nM, 10nM, 0.001mM, and 0.1mM as the same as the effect of different auxin concentrations experiment. These different auxin concentrations allow the roots to show different architectures. The experiments are planned up as shown in the diagrams below.
- The material for making the slope is the 20 cm x 10 cm pots placing on top of the 30 degree slope.
- The soil chosen is called M2 which composes of organic compost without any gravel to eliminate the error from different mixtures which contributes to different soil contents. The soil immitates normal
- The pressure of water is kept constant by the adaptable shower head which immitates the average speed of rainfall (9.8 m/s)

The experiment is set up where 18 (3 columns at 10 cm side x 6 rows on 20 cm side) arabidopsis are seeded on the pots which represents each concentration. At each concentration, the experiment is repeated 3 times. These altogether with 2 controls where no auxin is watered in the first one and no Arabidopsis is seeded in the second one, result in 30 pots in total. Estimately 12.5 kg of soil (1/4 of 50kg M2) is used for the experiment. The soil is pretreated with 2 litre of 1mM fungicide solution and left dryout for 1 day. It is then seperated to approximately 400g on each pot and is watered each with 25ml of water to moist up the soil conditions for Arabidopsis seeding. A small hole is digged onto each seeding place to fix the position of the seeds. Arabidopsis is seeded with the pattern already described. For the first 3 days, the seeds are watered everyday at 4 pm to promote the growth and the watering is decreased to once in a 2 days afterwards. Each pot is watered with 25 ml of auxin concentrations mentioned above.