Team:Imperial College London/Project Chemotaxis Overview

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<h1>Overview</h1>
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<h2>The module</h2>
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<p><i>Figure 1: Escherichia coli cells expressing superfolder GFP (sfGFP) can be seen inside an Arabidopsis thaliana root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "wrong positives" of bacteria adhering to the outside of the root. For a cool 3D video of bacteria inside the roots, check out our <a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Testing">Results</a> page. (Data and imaging by Imperial iGEM 2011).</i></p>
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<p><i>Figure 1: </i>Escherichia coli<i> cells expressing superfolder GFP (sfGFP) can be seen inside an </i>Arabidopsis thaliana<i> root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root. For a cool 3D video of bacteria inside the roots, check out our <a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Testing"><b>Results</b></a> page. (Data and imaging by Imperial iGEM 2011).</i></p>
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<h1>Overview</h1>
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<p>The Phyto-Route module mainly consists of bacterial movement towards plant roots. Following bacterial movement to the roots, the microbes are taken up into the roots themselves. The fact that bacteria are taken up into plant roots, where they are used for nutrients by the plant, is a novel finding that was only described last year when Paungfoo-Lonhienne et al. <sup>[1]</sup> reported the uptake of non-pathogenic <i>Escherichia coli</i> into the roots of <i>Arabidopsis thaliana</i> (watercress) and <i>Lycopersicum esculentum</i> (tomato plant). We have successfully replicated these findings (Figure 1). This is especially interesting for our project as the plants will be exposed to bacterial auxin inside the root cells, which significantly affects the concentration of IAA the bacteria need to produce to effect optimal root growth. In addition, Phyto-Route can potentially be used as a platform to deliver compounds, which would not naturally occur in the plant, into roots without genetically modifying the plant itself.</p>
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<p>The Phyto Route module mainly consists of bacterial movement towards plant roots. Following bacterial movement to the roots, the microbes will be taken up into the roots of the plants. The fact that bacteria are taken up into plant roots, where they are used for nutrients by the plant itself, is a novel finding that was only described last year. Paungfoo-Lonhienne et al. (1) described the uptake of <i>Escherichia coli</i> into the roots of watercress and tomato plants. We have replicated these findings (Figure 1). This is especially interesting for our project as the plants will be exposed to bacterial auxin inside the root cells.</p>
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<h2>Bacterial chemotaxis</h2>
<p>Our primary chassis for wet lab experiments is <i>Escherichia coli</i>. Chemotaxis in <i>E. coli</i> 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.</p>
<p>Our primary chassis for wet lab experiments is <i>Escherichia coli</i>. Chemotaxis in <i>E. coli</i> 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.</p>
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<p>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, with basic functioning having same as typical prokaryotic two component system.
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<p>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, please see the demonstration below for details <sup>[2]</sup>.</p>
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First part of the mechanism is sensory kinase, which consists of input domain and autokinase domain. Second part of the mechanism is the response regulator, with reciever and output domains. In the case of chemotactic system, sensory kinase is chemoreceptor associated with CheA and CheW proteins. This association remains present only in the absence of a ligand. During that period CheA autophosphorylates and is capable of phosphorylating CheY, protein which acts as a response regulator in this mechanism. Phosphorylated CheY has the capability of associating itself with flagellar proteins, thereby controlling the direction which flagellum rotates. However, in the presence of ligand, sensory kinase domain is not functional due to dissociation of CheA from chemoreceptor. This way CheY does nto associate with flagellar proteins and result is counterclockwise flagellar movement (Sourjik & Armitage, 2010).</p>
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<p>If you cannot see the Flash animation below, download the Adobe Flash Player <a href="http://www.adobe.com/support/flashplayer/downloads.html" target="_blank"><b>here</b></a>.</p>
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<p><embed src="https://static.igem.org/mediawiki/2011/5/54/ICL_Chemotaxis.swf" width="935px" height="500px" style="border:1px solid black;"/></p>
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<p> In <i>E. coli</i> chemotaxis there are a number of other proteins, which have functions associated with the two-component system and as a result they enable the bacterium to move up or down a concentration gradient. 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. This is driven by CheZ, a phosphatase that removes phosphate groups from CheY, while sensory kinase is dissociated. In addition, CheB  acts as a methylesterase and can remove methyl groups from the MCP receptor, to act as a memory reset (Chelsky & Dahlquist, 1980).</p>
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<h2>References</h2>
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<p>[1] Paungfoo-Lonhienne C et al. (2010) Turning the table: plants consume microbes as a source of nutrients. <i>PLoS One</i> <b>5(7):</b> e11915.</p>
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<p>[2] Chelsky D and Dahlquist FW (1980) Chemotaxis in <i>Escherichia coli</i>: association of protein components. <i>Biochemistry</i> <b>19:</b> 4633–4639.</p>
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<p><embed src="https://static.igem.org/mediawiki/igem.org/5/54/ICL_Chemotaxis.swf" width="935px" height="500px" style="border:1px solid black;"/></p>
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The Problem
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<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Chemotaxis_Specifications" style="text-decoration:none;color:#728F1D;float:right;">
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M1: Specifications
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Latest revision as of 23:09, 16 October 2011



Module 1: Phyto-Route

Chemotaxis is the movement of bacteria based on attraction or repulsion of chemicals. Roots secrete a variety of compounds that E. coli are not attracted to naturally. Accordingly, we engineered a chemoreceptor into our chassis that can sense malate, a common root exudate, so that it can swim towards the root. Additionally, E. coli are actively taken up by plant roots, which will allow targeted IAA delivery into roots by our system.





Overview

The module

Figure 1: Escherichia coli cells expressing superfolder GFP (sfGFP) can be seen inside an Arabidopsis thaliana root using confocal microscopy after overnight incubation of the plants with bacteria. Roots were washed in PBS prior to imaging to avoid "false positives" of bacteria adhering to the outside of the root. For a cool 3D video of bacteria inside the roots, check out our Results page. (Data and imaging by Imperial iGEM 2011).

The Phyto-Route module mainly consists of bacterial movement towards plant roots. Following bacterial movement to the roots, the microbes are taken up into the roots themselves. The fact that bacteria are taken up into plant roots, where they are used for nutrients by the plant, is a novel finding that was only described last year when Paungfoo-Lonhienne et al. [1] reported the uptake of non-pathogenic Escherichia coli into the roots of Arabidopsis thaliana (watercress) and Lycopersicum esculentum (tomato plant). We have successfully replicated these findings (Figure 1). This is especially interesting for our project as the plants will be exposed to bacterial auxin inside the root cells, which significantly affects the concentration of IAA the bacteria need to produce to effect optimal root growth. In addition, Phyto-Route can potentially be used as a platform to deliver compounds, which would not naturally occur in the plant, into roots without genetically modifying the plant itself.


Bacterial chemotaxis

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, please see the demonstration below for details [2].


If you cannot see the Flash animation below, download the Adobe Flash Player here.

References

[1] Paungfoo-Lonhienne C et al. (2010) Turning the table: plants consume microbes as a source of nutrients. PLoS One 5(7): e11915.

[2] Chelsky D and Dahlquist FW (1980) Chemotaxis in Escherichia coli: association of protein components. Biochemistry 19: 4633–4639.

The Problem M1: Specifications



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