Team:Dundee/Project/Mop

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           <h2><b>Toxi Mop </b> - Splash and the toxin's gone!</h2>
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           <h2><b>The Mop</b> </h2>
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          <h2>Aim: To engineer the <i> B. subtilis</i>  receptor PrkC to respond to microcystin</h2>
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          <p> <i> B. subtilis</i> forms resistant structures called spores in order to survive harsh environmental conditions. In order for the spores to recognise that the conditions have again become favourable for growth the spores have to monitor the extracellular environment. This is done via a number of inner-membrane receptors described as germinant receptors. PrkC is an example of a germinant receptor and it binds to cell wall associated peptides.</p>
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          <h2>Sensing cell wall peptides & conditions that are permissive for growth </h2>
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          <p> Actively growing cells turnover cell wall components and these can thus be found in the extracellular milieu. So by sensing cell wall components, through the PrkC receptor, the spore can tell that other cells are growing in the nearby environment. This is how the PrkC receptor can signal to the spore that conditions are permissive for growth.</p><br>
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        <h2>PrkC receptor activation</h2>
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        <p>PrkC receptor activation triggers a process called germination which is the conversion of the spore back into an actively growing cell.</p><br>
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        <h2>PrkC receptor</h2>
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        <p>The PrkC receptor has 4 extracellular domains PASTA 1, 2 and 3 which are capped by a C-terminal domain and this sits on the outside of the spore inner membrane. The 3 PASTA domains are implicated in binding of the cell wall components and are thus described as the ligand binding domains (fig 1).</p><br>
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        <h2>PrkC receptor to detect microcystin</h2>
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        <p>We hope to detect microcystin by replacing the 3 ligand binding domains with the human protein- protein phosphatase 1 (PP1) (fig 2).</p><br>
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        <p>We hope that when the microcystin binds to PP1 this will still result in activation of the downstream pathways controlled by the native PrkC receptor. Additionally, we hope to have our <i> B. subtilis</i> strain constitutively expressing green fluorescent protein so that when it is relieved from dormancy it will fluoresce and this will hopefully be detectable with our electronic Moptopus device.</p>
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        <h2 style="margin-top:-10px;"> The Microcystin Monster </h2>
 
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        <p> Algal blooms are an ever-growing problem in freshwater systems. At the Beijing Olympics 2008, 10,000 people were hired to clean up the extensive algal bloom in time for the sailing regatta. The main concern is the level of a toxin called microcystin, which is released by cyanobacteria when they die and lyse. <br><br>Currently, the method of detection takes a day to produce results, so our aim as a team is to develop a 60 minute microcystin detection system, as well as a method to combat the rising levels of the toxin in lakes, ponds, etc. The iGEM Dundee team were inspired to act on this problem due to not only its effect on the local freshwater reservoirs, but worldwide. </p>
 
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          <br> <p><i><b>Figure 1.</b> .</i></p><br>
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            <p><br><i>Figure 2. .</i></p>
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        <h2>Progress so far...</h2>
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        <p>We are currently in the process of cloning this receptor and we are having some difficulty. We have successfully cloned the N-terminal part of the receptor and are currently in the process of adding on the PP1s which we will be doing by suicide ligation. The final step after adding the PP1s will be to add on the C-terminal domain. </p><br><br>
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          <h2 style="margin-top:-10px;"> Save the Janitor, Save the world! </h2>
 
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          <p> Microcystin, a toxin released by Microcystis aeruginosa, is harmful to mammals due to its ability to latch on to the human protein PP1, thus ceasing its operation.
 
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          We are exploiting the ability of the human protein phosphatase (PP1) to covalently bind to microcystin, in order to develop a biological mop ‘janitor’ to rid algal bloom water of the toxin. <br><br>By changing domains on receptors on the cell surface of e.coli and b.subtilis, we plan to develop a method of microcystin detection. Thirdly, iGEM Dundee are creating ‘Moptopus’; a remote environmental monitoring device which is designed to detect pH, temperature, light, dissolved oxygen in H2O and even has a robotic eye. Moptopus can be controlled online and can even send tweets to alert the public whenever an algal bloom is imminent.
 
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        <h2 style="margin-top:-10px;"> Unmasking the Monster </h2>
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        The public generally considers synthetic biology as an immoral concept, although if you imagine it as an episode of Scooby Doo, it doesn’t seem so bad; everyone is scared of this unknown monster, but underneath this mask is just a janitor. In the case of our project ToxiMop, we are using a ‘janitor bacterium’ to mop up the microcystin toxin from freshwater reservoirs!
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          <h2>Aim: To engineer the <i>E. coli</i> EnvZ sensor kinase to respond to microcystin</h2>
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          <p>The EnvZ system is a signal transduction system composed of two parts and is, therefore, described as a two-component regulatory system. Part 1 is the sensor kinase protein located in the membrane of the cell and Part 2 is the response regulator protein. The native EnvZ sensor detects changes in osmolarity. </p>
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          What comes to people's mind when they hear the term 'synthetic biology'? Many people don't know what it is, or have an ambiguous idea that it is something dangerous, potentially immoral. It can be thought of as playing with the universe's lego kit. Building with what is already here, naturally, biologists attempt to create better biological systems and machinery to advance life on earth. </p>
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        <h2>EnvZ sensor kinase</h2>
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          <p> The sensor kinase EnvZ detects a signal from the environment and auto-phosphorylates. The phosphoryl group is then transferred to the response regulator OmpR. OmpR is a DNA-binding protein.
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          E.coli is a gram-negative bacteria which means that is has both an inner and outer membrane. The EnvZ sensor sits on the inner membrane (Fig 3).
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        <h2>EnvZ sensor to detect microcystin</h2>
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        <p>What we want to do for our project is replace the periplasmic domain of EnvZ with the PP1 protein (Fig 4). We hope that when microcystin binds to PP1 then it will activate the receptor.</p><br>
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        <p>This will lead to the phosphorylation and activation of the DNA binding protein OmpR. We will also express in our engineered bacteria a DNA construct encoding GFP that’s expression is under control of the OmpR protein.<br><br> So our cells will turn green in the presence of microcystin and in this way act as a microcystin detector. </p>
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                          <p><b style="font-size:16px;">Toxi-Mop</b><br><br> We are using cloning techniques to genetically engineer B. subtilis and E. coli to express PP1 so that they can inhibit the toxin microcystin in algal blooms, therefore reducing harm to freshwater ecosystems. ”</p>
 
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                          <p><b style="font-size:16px;">Project Mop-topus</b><br><br>  A remotly accessed electronic environmental sensor that detects and monitors the state of a lake and its susceptibility to algal blooms by measuring light, temperature, pH, and dissolved oxygen variables.</p>
 
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                          <p><b style="font-size:16px;">The Detector</b><br><br> We are making 2 different microcystin detectors by substituting domains of bacterial cell surface receptors involved with gene regulation, with PP1 molecules. </p>
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          <br> <p><i><b>Figure 3.</b> .</i></p><br>
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            <p><br><i>Figure 4. .</i></p>
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        <h2>Progress so far...</h2>
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        <p>WSo far we have successfully cloned the N-terminus with PP1 and we are in the process of adding on the C-terminus. We have also found an OmpR regulated construct in the distribution kit and we have transformed cells to make more of this part. We have also identified GFP in the kit and we will try and join these 2 parts together to make our reporter construct.
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        <h2>Characterisation of our receptors</h2>
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        <p>We will be looking to quantify how many of our PrkC receptors are expressed on the surface of the spores and also how many EnvZ sensors are present on our <i>E. coli</i> cells.<br><br>
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        We will be able to get hold of some microcystin and we can use this to bind and activate our receptors. We will then measure the amount of fluorescence by flow cytometry or microscopy. We can then quantify the expression of GFP in relation to how much microcystin is presented to our cells. By using values for how many receptors we have on each cell we can calculate the efficiency of our detectors and hopefully use all this information in order to quantify the effectiveness of our detector.
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                          <p><b style="font-size:16px;">Our Team</b><br><br>The team is consists of biologists, a mathematician, a math biologist, a physicist and a software engineer. By bringing together students with different expertise, we strive to maintain and improve upon previous iGEM teams' achievements.</p>
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Revision as of 11:08, 10 July 2013

iGEM Dundee 2013 · Toxi-Mop

Dundee iGEM 2013

The Mop

Aim: To engineer the B. subtilis receptor PrkC to respond to microcystin

B. subtilis forms resistant structures called spores in order to survive harsh environmental conditions. In order for the spores to recognise that the conditions have again become favourable for growth the spores have to monitor the extracellular environment. This is done via a number of inner-membrane receptors described as germinant receptors. PrkC is an example of a germinant receptor and it binds to cell wall associated peptides.

Sensing cell wall peptides & conditions that are permissive for growth

Actively growing cells turnover cell wall components and these can thus be found in the extracellular milieu. So by sensing cell wall components, through the PrkC receptor, the spore can tell that other cells are growing in the nearby environment. This is how the PrkC receptor can signal to the spore that conditions are permissive for growth.


PrkC receptor activation

PrkC receptor activation triggers a process called germination which is the conversion of the spore back into an actively growing cell.


PrkC receptor

The PrkC receptor has 4 extracellular domains PASTA 1, 2 and 3 which are capped by a C-terminal domain and this sits on the outside of the spore inner membrane. The 3 PASTA domains are implicated in binding of the cell wall components and are thus described as the ligand binding domains (fig 1).


PrkC receptor to detect microcystin

We hope to detect microcystin by replacing the 3 ligand binding domains with the human protein- protein phosphatase 1 (PP1) (fig 2).


We hope that when the microcystin binds to PP1 this will still result in activation of the downstream pathways controlled by the native PrkC receptor. Additionally, we hope to have our B. subtilis strain constitutively expressing green fluorescent protein so that when it is relieved from dormancy it will fluoresce and this will hopefully be detectable with our electronic Moptopus device.



Figure 1. .





Figure 2. .

Progress so far...

We are currently in the process of cloning this receptor and we are having some difficulty. We have successfully cloned the N-terminal part of the receptor and are currently in the process of adding on the PP1s which we will be doing by suicide ligation. The final step after adding the PP1s will be to add on the C-terminal domain.



Aim: To engineer the E. coli EnvZ sensor kinase to respond to microcystin

The EnvZ system is a signal transduction system composed of two parts and is, therefore, described as a two-component regulatory system. Part 1 is the sensor kinase protein located in the membrane of the cell and Part 2 is the response regulator protein. The native EnvZ sensor detects changes in osmolarity.

EnvZ sensor kinase

The sensor kinase EnvZ detects a signal from the environment and auto-phosphorylates. The phosphoryl group is then transferred to the response regulator OmpR. OmpR is a DNA-binding protein.

E.coli is a gram-negative bacteria which means that is has both an inner and outer membrane. The EnvZ sensor sits on the inner membrane (Fig 3).


EnvZ sensor to detect microcystin

What we want to do for our project is replace the periplasmic domain of EnvZ with the PP1 protein (Fig 4). We hope that when microcystin binds to PP1 then it will activate the receptor.


This will lead to the phosphorylation and activation of the DNA binding protein OmpR. We will also express in our engineered bacteria a DNA construct encoding GFP that’s expression is under control of the OmpR protein.

So our cells will turn green in the presence of microcystin and in this way act as a microcystin detector.



Figure 3. .





Figure 4. .

Progress so far...

WSo far we have successfully cloned the N-terminus with PP1 and we are in the process of adding on the C-terminus. We have also found an OmpR regulated construct in the distribution kit and we have transformed cells to make more of this part. We have also identified GFP in the kit and we will try and join these 2 parts together to make our reporter construct.

Characterisation of our receptors

We will be looking to quantify how many of our PrkC receptors are expressed on the surface of the spores and also how many EnvZ sensors are present on our E. coli cells.

We will be able to get hold of some microcystin and we can use this to bind and activate our receptors. We will then measure the amount of fluorescence by flow cytometry or microscopy. We can then quantify the expression of GFP in relation to how much microcystin is presented to our cells. By using values for how many receptors we have on each cell we can calculate the efficiency of our detectors and hopefully use all this information in order to quantify the effectiveness of our detector.



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