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	<h2>Testing</h2>		<h2>Testing</h2>
-	<p>	+	<p>Testing for chemotaxis can be split into qualitative and quantitative assays. Qualitative assays involve putting engineered <i>E. coli</i> and an attractant onto semi-solid agar plates and observe the movement of the microbes. If they can be observed to move towards the attractant source, they are likely to be attracted to the ligand. In quantitative assays, capillaries are filled with different concentrations of the attractant malate. Positive controls are provided by filling identical capillaries with different concentrations of serine, which <i>E. coli</i> naturally move towards. Negative controls are provided by filling capillaries with media that does not contain a source of attractant. The amount of bacteria that swim into each capillary is evaluated by FACS.</p>
	<p> For simplicity, we will be working with Arabidopsis to observe the uptake of bacteria into plant roots.		<p> For simplicity, we will be working with Arabidopsis to observe the uptake of bacteria into plant roots.

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Revision as of 10:28, 25 August 2011

Chemotaxis

Movement performed by bacteria based on attraction or repulsion of chemicals is known as chemotaxis. In our project we are using this mechanism for translocation to plant roots of our modified microbes. They will be attracted to the root and will actively swim towards it.

Our primary chassis for wet lab experiments is Escherichia coli. Chemotaxis in E. coli is well documented. These bacteria can perform two types of movement, tumbling and smooth swimming. The difference between the two is determined by flagellar movement. During tumbling movement, the flagella move clockwise. This is caused by the formation of a complex between CheY-P and FliM, one of the flagella-associated proteins. During smooth swimming, the flagella move counter-clockwise. CheY is not phosphorylated and therefore cannot associate with flagellar proteins, causing the flagella to rotate in the opposite direction.

Smooth swimming is the movement performed by bacteria towards an attractant or away from a repellent. Smooth swimming is controlled by a number of chemotaxis proteins that make up a signalling pathway. At the start of the signalling pathway there is a receptor that binds to CheW and CheA in the absence of ligands (attractants or repllents). This complex phosphorylates CheY, which then associates with flagellar proteins. When a ligand binds to the receptor, CheW and CheA dissociate from the receptor. Subsequently, they are unable to phosphorylate CheY, rendering CheY unable to bind FliM. This leads to counterclockwise flagellar movement.

As part of chemotaxis, bacteria display memory. It enables them to move up or down a concentration gradient. This is driven by CheZ, a phosphatase that removes phosphate groups from CheY. This is mediated by CheR, a methyltransferase that methylates MCP (methyl accepting chemotaxis protein). This affects the receptor’s ability to associate with CheW and CheA. Dissociation of CheW and CheA from the chemoreceptor depends on the rising concentration of attractant, which in turn depends on the bacterium moving towards the source of attraction. In addition, CheB acts as a methylesterase and can remove methyl groups from the MCP receptor (Chelsky & Dahlquist, 1980).

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Specification

The chemotaxis module is responsible for ensuring that our bacteria move towards roots. For this, the bacteria need to be able to sense malate, which is secreted by roots. Subsequently, they need to be able to move towards malate. E. coli, the chassis we are using for lab experiments, does not normally exhibit chemotaxis towards malate. Accordingly, we needed to engineer a malate-responsive sensor into the microbes that will enable them to perform chemotaxis towards roots.

Following chemotaxis towards the roots, our bacteria should be taken up into the roots. We want the bacteria to get taken up into the plant roots to ensure that the concentration of indole-3-acetic acid in the plant is increased. If the bacteria remained outside the roots, this goal may also be reached but the risk that we would not increase the internal IAA concentration would be significantly higher. In addition, uptake of bacteria into the roots followed by secretion of chemicals presents a novel platform for modifying plants without engineering their genomes. In a paper published last year, Paungfoo-Lonhienne et al. showed that Arabidopsis and tomato plants are able to actively break down their cell wall to take up GFP-tagged E. coli and S. cerevisiae and use them as a source of nutrients.

Design

While malate-responsive sensors do not occur in E. coli, they have been identified in several other bacteria species, including the soil microbe Pseudomonas putida. We therefore chose to engineer the chemoreceptor mcpS from the P. putida KT2440 strain. McpS is a receptor that responds to a number of TCA cycle intermediates such as malate, fumarate, oxaloacetate, succinate, citrate, isocitrate and butyrate (Lacal et al, 2010). P. putida and E. coli use different proteins for chemotaxis. However, these proteins are structurally similar and it is reasonable to assume that the mcpS domain will interact with the Che proteins in E. coli and the bacteria will be able to translocate upon malate binding. At the same time, we are utilising the chemotaxis receptor PA2652 from the Pseudomonas aeruginosa PA01 strain, which also uses malate as attractant ligand. This approach will enable us to determine which of the two receptors is more responsive.

To ensure that the genes will be expressed optimally, the genes for the receptors were codon-optimised for both E. coli and Bacillus subtilis, the two chassis we are working with. We wrote a codon optimisation program for this purpose.

In addition, we chose a 15bp insulator sequence between the RBS and the promoter. This sequence was specifically designed to make the promoter interchangeable without affecting the RBS and thus improve modularity of the receptor. The insulator sequence acts as a starting point for PCR and it can be used to amplify the receptor sequence without the promoter.

We will demonstrate that foreign chemoreceptors are compatible with the E. coli chemotaxis system, provided the parts share substantial structural similarity. In addition, our approach will demonstrate that it is possible to increase the number of attractants to which E. coli is attracted by adding compatible chemoreceptors.

Root uptake of both E. coli and the bakers’s yeast S. cerevisiae can be observed in the model organism Arabidopsis thaliana and in tomato plants. It is a process that occurs naturally (althought it yet remains to be observed in soil) and we do not need to incorporate additional genes into our design.

Modelling

Two main aspects were modelled for our chemotaxis module: malate distribution in soil and the threshold concentration of malate needed to trigger chemotaxis.

The malate concentration distribution was modelled using the Keller-Segel model.

Assembly

The receptor genes were synthesised in two fragments. In order to assemble the construct, we -use CPEC to combine the two fragments

Testing

Testing for chemotaxis can be split into qualitative and quantitative assays. Qualitative assays involve putting engineered E. coli and an attractant onto semi-solid agar plates and observe the movement of the microbes. If they can be observed to move towards the attractant source, they are likely to be attracted to the ligand. In quantitative assays, capillaries are filled with different concentrations of the attractant malate. Positive controls are provided by filling identical capillaries with different concentrations of serine, which E. coli naturally move towards. Negative controls are provided by filling capillaries with media that does not contain a source of attractant. The amount of bacteria that swim into each capillary is evaluated by FACS.

For simplicity, we will be working with Arabidopsis to observe the uptake of bacteria into plant roots. Arabidopsis thaliana is a common plant model organism. It belongs to the mustard family and fulfils many important requirements for model organisms. As such, its genome has been almost completely sequenced and replicates quickly, producing a large number of seeds. It is easily transformed and many different mutant strains have been constructed to study different aspects (National Institute of Health, no date). While Arabidopsis may not represent plant populations naturally occurring in arid areas threatened by desertification, it is a handy model organism we will be using to study the effect of auxin on roots, observe chemotaxis towards them and look at uptake of bacteria into the roots. We will be using Arabidopsis to look at the uptake of our engineered bacteria into the plants. For this, we will be using wild type Arabidopsis and E. coli that constitutively express green fluorescent protein. The natural fluorescence produced by plant roots and green fluorescence produced by the bacteria can be used to image the uptake of bacteria using confocal microscopy.

References

Chelsky, D. & Dahlquist, F. W. (1980) Chemotaxis in Escherichia coli: Association of protein components. Biochemistry, 19, 4633 – 4639.
Lacal, J., Alfonso, C., Liu, X. & et al. (2010) Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands. Journal of Biological Chemistry, 285 (30), 23126 – 23136.
Paungfoo-Lonhienne et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS ONE 5(7): e11915. http://www.nih.gov/science/models/arabidopsis/index.html