Team:Imperial College London/Project/Auxin/Overview

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<p>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).</p>
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<h1>Overview</h1>
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<p>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.</p>
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<h2>Design:</h2>
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<p>Auxin is a well known plant hormone that is responsible for plant growth in response to biotic and abiotic stresses. Usually, synthetic auxins like α-Naphthaleneacetic acid (αNAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) are used as herbicides. They have been been used effectively for the past fifty years due to their high effectiveness and cheap cost. This high efficiency stems from the interactions that these auxin analogues have with the TIR1 protein. When the analogues bind to the TIR1 protein promoting the formation of the Aux/IAA–SCFTIR1. This leads to the ubiquitylation of of Aux/IAA which usually acts as an inhibitor when bound to ARF transcription factors. Many of the plant's genes are under the control of the ARF transcription factor meaning that any alteration can have a large impact on the plant's morphology. When altered by persistent compounds that cannot be degraded easily (such as synthetic auxins), the plant suffers as it is not able to keep up with the high metabolic demand correlated with over-expression of genes.</p>
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<p>IaaM and IaaH have been codon optimized for both Bacillus subtilis and E. coli through the use of our own codon optimizing software. Also, the genes have been placed under the pVEG promoter which works in B. subtilis and E. coli and we calculated the RBS efficiency for both E. coli and B. subtilis. Furthermore, insulator sequences have been placed in front of the ribosome binding sites so that the genes could be placed under different promoters depending on desired output in different species.</p>
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<p>Therefore, we believe that natural auxin will not function as a herbicide because it is a labile chemically that can be easily metabolized by the plants. However, there is still a danger that too much natural auxin can also be detrimental to the plants. Therefore, for this module we must consider this fact during our design process. The bacteria must be able to produce a certain amount of auxin and must not go above a certain threshold. This level of control makes it a perfect project for synthetic biology which is looking to design controllable components in natural systems.</p>
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<p>Our specifications will therefore be influenced by our modelling as well as our experiments on <i>Arabidopsis thaliana</i>. These experiments include an auxin concentration assay where we will be looking at the effect of auxin on the root length as well as the total dry mass of the plant in phytogel and soil. We will also be looking through past literature in order to determine what concentrations of auxin are known to promote root growth. However, seeing as how we only have ten weeks for the project and these sorts of studies take a long time, we must create a construct that will be able to produce auxin and will be as modular as possible to allow for easy tinkering in later stages. We have decided to use the IAM pathway from <i>Pseudomonas savastanoi</i> since it has been shown to be expressed in <i>Escherichia coli</i> and is only composed of two enzymes.</p>
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<img src=https://static.igem.org/mediawiki/2011/c/cd/ICL_Auxin_Module_cell.png width=700px/>
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<p>For the first design of our Auxin Xpress module we decided to make it as modular as possible. We have introduced an insulator sequence which will allow us to test out different promoters in the future until we find a combination of components that will fit our specifications. We also decided to use a variant of the Pveg promoter to contribute further to our modularity as well as codon optimizing our sequences for both <i>Bacillus subtilis</i> and <i>Escherichia coli</i>. This flexibility allowed us to choose our chassis by calmly looking at the pros and cons of both bacteria. We came to the conclusion that <i>E. coli</i> would be a better chassis due to its ease of production as well as its inability to form spores which would make the issue of containment a nightmare (seeing as how we would be working with loose soil onto which the spores could attach themselves to and be blown far distances from the application zone).</p>
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<h2>The IAM pathway:</h2>
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<p>Our modelling has looked into the rate constants of the two enzymes in our system (IaaM and IaaH) in order to calculate how much of the enzymes we would need to produce to create the desired output. However, in order to more accurately determine whether the equations give us a correct output we must first look at how much auxin our first version of this module will produce. The desired output has been modelled by using the data from the auxin effect experiments on <i>A. thaliana</i> through a process known as data fitting. Therefore, our modelling will allow us to calculate how many cells we would require on each seed or what promoter and RBS might be more suitable if a second version of our construct were to be built.</p>
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<p>The IAM pathway taken for this module is from Pseudomonas savastanoi. This strain of soil dwelling bacteria is a known plant pathogen that uses IAA to infect its target. However, there have been some recent studies that suggest that IAA secretion by bacteria can also lead to positive microb-plant relations [1]. Therefore, we must carefully analyze what IAA concentration would aid root growth rather than promote gall formation. To achieve this, we will be experimenting with different levels of synthetic auxin on Arabidopsis thaliana. We will also be modelling this module in order to obtain the adequate concentration of IAA excretion from the chassis.</p>
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<h2>The effect on plants</h2>
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<p>The first version of our module was built through the use of our new standard. Our new standard consists of ordering synthetically made DNA fragments with 50bp overlaps that are flanked by MlyI restriction sites. This allows for the easy blunt excision of the fragments from their expression vectors. Once the fragments have been excised one can put them together through the use of Gibson (making it a PCR-less Gibson) or CPEC. For this construct we used CPEC.</p>
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<p>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. </p>
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<p>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. </p>
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<p>Finally, once the module was assembled we decided to use a simple colourimetric assay with Salkowski reagent. Salkowski reagent is commonly used in order to determine a rough estimate for the amount of Auxin present within a cell culture filtrate. If auxin is present, the reagent turns pinkish-red. For a more accurate analysis on the amount of auxin we are producing we are going to use HPLC with a reverse phase C18 column. Once these two assays are completed we fed the data back through the modelling and determined that </p>
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<p>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.</p>
 
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<p>References:</p>
 
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[1] Stijn Spaepen, Jos Vanderleyden, and Roseline Remans, “Indole-3-acetic acid in microbial and microorganism-plant signaling,” FEMS Microbiology Reviews 31, no. 4 (July 2007): 425-448.
 
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Latest revision as of 01:18, 15 September 2011



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.



Overview

Auxin is a well known plant hormone that is responsible for plant growth in response to biotic and abiotic stresses. Usually, synthetic auxins like α-Naphthaleneacetic acid (αNAA) and 2,4-Dichlorophenoxyacetic acid (2,4-D) are used as herbicides. They have been been used effectively for the past fifty years due to their high effectiveness and cheap cost. This high efficiency stems from the interactions that these auxin analogues have with the TIR1 protein. When the analogues bind to the TIR1 protein promoting the formation of the Aux/IAA–SCFTIR1. This leads to the ubiquitylation of of Aux/IAA which usually acts as an inhibitor when bound to ARF transcription factors. Many of the plant's genes are under the control of the ARF transcription factor meaning that any alteration can have a large impact on the plant's morphology. When altered by persistent compounds that cannot be degraded easily (such as synthetic auxins), the plant suffers as it is not able to keep up with the high metabolic demand correlated with over-expression of genes.

Therefore, we believe that natural auxin will not function as a herbicide because it is a labile chemically that can be easily metabolized by the plants. However, there is still a danger that too much natural auxin can also be detrimental to the plants. Therefore, for this module we must consider this fact during our design process. The bacteria must be able to produce a certain amount of auxin and must not go above a certain threshold. This level of control makes it a perfect project for synthetic biology which is looking to design controllable components in natural systems.

Our specifications will therefore be influenced by our modelling as well as our experiments on Arabidopsis thaliana. These experiments include an auxin concentration assay where we will be looking at the effect of auxin on the root length as well as the total dry mass of the plant in phytogel and soil. We will also be looking through past literature in order to determine what concentrations of auxin are known to promote root growth. However, seeing as how we only have ten weeks for the project and these sorts of studies take a long time, we must create a construct that will be able to produce auxin and will be as modular as possible to allow for easy tinkering in later stages. We have decided to use the IAM pathway from Pseudomonas savastanoi since it has been shown to be expressed in Escherichia coli and is only composed of two enzymes.

For the first design of our Auxin Xpress module we decided to make it as modular as possible. We have introduced an insulator sequence which will allow us to test out different promoters in the future until we find a combination of components that will fit our specifications. We also decided to use a variant of the Pveg promoter to contribute further to our modularity as well as codon optimizing our sequences for both Bacillus subtilis and Escherichia coli. This flexibility allowed us to choose our chassis by calmly looking at the pros and cons of both bacteria. We came to the conclusion that E. coli would be a better chassis due to its ease of production as well as its inability to form spores which would make the issue of containment a nightmare (seeing as how we would be working with loose soil onto which the spores could attach themselves to and be blown far distances from the application zone).

Our modelling has looked into the rate constants of the two enzymes in our system (IaaM and IaaH) in order to calculate how much of the enzymes we would need to produce to create the desired output. However, in order to more accurately determine whether the equations give us a correct output we must first look at how much auxin our first version of this module will produce. The desired output has been modelled by using the data from the auxin effect experiments on A. thaliana through a process known as data fitting. Therefore, our modelling will allow us to calculate how many cells we would require on each seed or what promoter and RBS might be more suitable if a second version of our construct were to be built.

The first version of our module was built through the use of our new standard. Our new standard consists of ordering synthetically made DNA fragments with 50bp overlaps that are flanked by MlyI restriction sites. This allows for the easy blunt excision of the fragments from their expression vectors. Once the fragments have been excised one can put them together through the use of Gibson (making it a PCR-less Gibson) or CPEC. For this construct we used CPEC.

Finally, once the module was assembled we decided to use a simple colourimetric assay with Salkowski reagent. Salkowski reagent is commonly used in order to determine a rough estimate for the amount of Auxin present within a cell culture filtrate. If auxin is present, the reagent turns pinkish-red. For a more accurate analysis on the amount of auxin we are producing we are going to use HPLC with a reverse phase C18 column. Once these two assays are completed we fed the data back through the modelling and determined that

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