Team:Dundee/Project/Detector

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           <h2>Aim: To engineer the <i> B. subtilis</i> receptor PrkC to respond to microcystin</h2>
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           <h2>The detection systems</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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We designed two systems to detect microcystin, one in each of our chassis organisms.<br><br>
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<h2>Engineering the <i>B. subtilis</i> PrkC receptor to respond to microcystin</h2>
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<i>B. subtilis</i> forms desiccation-resistant structures called spores in order to survive harsh environmental conditions. In order for spores to know that the conditions have become favourable for germination and growth they must monitor the extracellular environment. This is achieved through 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.<br><br>
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<h2>Why sense cell wall peptides? How does this indicate that conditions are permissive for growth?</h2>
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Actively growing cells turnover cell wall components and these are released into 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 triggers a process called germination, which is the conversion of the spore back into an actively growing cell.<br><br>
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<h2>The PrkC receptor</h2>
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The extracellular portion of the PrkC receptor has 4 domains. Three of these are PASTA domains which are capped by a forth, C-terminal domain. The protein is anchored in the inner membrane and has an N-terminal kinase domain that phosphorylates downstream targets upon receptor activation. The 3 PASTA domains are implicated in binding of the cell wall components and are thus described as ligand binding domains (Fig 1A).<br><br>
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<h2>But how can we use the PrkC receptor to detect microcystin?</h2>
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We hope to detect microcystin by replacing the 3 ligand binding domains with three copies of PP1 (Fig 1B).<br><br>
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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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<center><img src="http://farm8.staticflickr.com/7292/10034973665_c91f4f9ea7_o.jpg"><br></center>
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<strong>Figure 1. : The <i>B. subtilis</i> PrkC Receptor (A) and the engineered receptor designed to respond to microcystin (B). </strong><br><br>
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We hope that when microcystin binds to the PP1 regions of the modified PrkC receptor this will result in activation of the downstream pathways controlled by native PrkC. Additionally, we aim to have our <i>B. subtilis</i> strain constitutively expressing GFP so that when it is relieved from dormancy it will fluoresce and this will be detectable with our electronic <a href="https://2013.igem.org/Team:Dundee/Project/SoftwareTheory" target="_blank">Moptopus device</a>.<br><br>
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<h2>Progress so far</h2>
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We are currently in the process of cloning this receptor and we are having some difficulty. We have successfully cloned the 5’ part of <i>prkC</i>, encoding the kinase domain, and are currently in the process of sequentially adding the PP1 genes by suicide ligation. The final step after this will be to ligate the 3’ end of <i>prkC</i>.<br><br>  
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        <h2>PrkC receptor activation</h2>
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<h2>Engineering the <i>E. coli</i> EnvZ sensor kinase to respond to microcystin</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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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 cell envelope and Part 2 is the cytoplasmic response regulator protein. The native EnvZ sensor detects changes in osmolarity.<br><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>EnvZ sensor kinase</h2>
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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. <i>E. coli</i> is a Gram-negative bacterium which means that it has inner and outer membranes. The EnvZ sensor sits on the inner membrane (Fig 2).<br><br>
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          <br> <p><i><b>Figure 1.</b> .</i></p><br>
 
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              <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Figure 2">
 
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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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<center><img src="http://farm3.staticflickr.com/2859/10035077703_80c60c236e_o.jpg"></center><br>
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<strong>Figure 2. The Native <i>E. coli</i> EnvZ Receptor.</strong> The N- and C-termini of EnvZ are located in the cytoplasm, with two transmembrane domains separated by a periplasmic loop. The periplasmic loop senses membrane tension caused by osmotic stress. This tension is transmitted the cytoplasmic side of the protein and triggers auto-phosphorylation.<br><br>
 +
<h2>EnvZ sensor to detect microcystin</h2>
 +
We want to replace the periplasmic domain of EnvZ with the PP1 protein (Fig 3), so that when microcystin binds to PP1 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 the GFP gene under control of the ompC promoter. This promoter is recognised and activated by phosphorylated OmpR and as a result, cells will turn green in the presence of microcystin, this acting as a microcystin detector.<br><br>
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<center><img src="http://farm8.staticflickr.com/7415/10035089443_977abb0772_o.jpg"></center><br>
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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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<strong>Figure 3. Schematic representation of the engineered EnvZ microcystin detector.</strong> In the engineered construct PP1 replaces the periplasmic domain of EnvZ.<br><br>
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<h2>Progress</h2>
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So far we have successfully cloned the 5’ and 3’ parts of </i>envZ</i>, replacing DNA encoding the periplasmic loop with that of the PP1 gene. Although this construct has been verified by sequencing, to date our attempts to express this hybrid protein have been unsuccessful. A possible reason for this is that the hybrid receptor is not correctly assembled. The periplasmic part of the receptor is translocated across the membrane by the Sec pathway, and we have already seen in our <a href="https://2013.igem.org/Team:Dundee/Project/MopMaking" target="_blank">mop experiments</a> that PP1 cannot be transported by Sec (possibly due to the presence of 13 cysteine residues in PP1 that may be aberrantly disulphide-bonded after translocation). It may be possible to overcome these limitations by re-engineering our hybrid EnvZ to interact with the Tat rather than the Sec pathway.
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The reporter under the control of OmpR has successfully been constructed, and <a href="https://2013.igem.org/Team:Dundee/Project/ReporterOmpC" target="_blank">confirmed to respond to OmpR by a change in expression of GFP.</a><br><br>
 +
<h2>Characterisation of our receptors</h2>
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We ultimately want 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 have purchased microcystin to test our mop, and we can use this to bind and activate our receptors. We will then measure the amount of fluorescence by flow cytometry or microscopy.  Then we can quantify the expression of GFP in relation to how much microcystin is presented to our cells. Using the number of receptors expressed in the membrane/spore for each cell we can calculate the effectiveness of our detector.
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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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          <br><br>
 
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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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          </p><br>
 
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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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          <br> <p><i><b>Figure 3.</b> .</i></p><br>
 
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              <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Figure 4">
 
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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><br><br>
 
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         </div>
          
          

Latest revision as of 10:56, 2 October 2013

iGEM Dundee 2013 · ToxiMop

The detection systems

We designed two systems to detect microcystin, one in each of our chassis organisms.

Engineering the B. subtilis PrkC receptor to respond to microcystin

B. subtilis forms desiccation-resistant structures called spores in order to survive harsh environmental conditions. In order for spores to know that the conditions have become favourable for germination and growth they must monitor the extracellular environment. This is achieved through 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.

Why sense cell wall peptides? How does this indicate that conditions are permissive for growth?

Actively growing cells turnover cell wall components and these are released into 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 triggers a process called germination, which is the conversion of the spore back into an actively growing cell.

The PrkC receptor

The extracellular portion of the PrkC receptor has 4 domains. Three of these are PASTA domains which are capped by a forth, C-terminal domain. The protein is anchored in the inner membrane and has an N-terminal kinase domain that phosphorylates downstream targets upon receptor activation. The 3 PASTA domains are implicated in binding of the cell wall components and are thus described as ligand binding domains (Fig 1A).

But how can we use the PrkC receptor to detect microcystin?

We hope to detect microcystin by replacing the 3 ligand binding domains with three copies of PP1 (Fig 1B).


Figure 1. : The B. subtilis PrkC Receptor (A) and the engineered receptor designed to respond to microcystin (B).

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

Progress so far

We are currently in the process of cloning this receptor and we are having some difficulty. We have successfully cloned the 5’ part of prkC, encoding the kinase domain, and are currently in the process of sequentially adding the PP1 genes by suicide ligation. The final step after this will be to ligate the 3’ end of prkC.

Engineering 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 cell envelope and Part 2 is the cytoplasmic 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 bacterium which means that it has inner and outer membranes. The EnvZ sensor sits on the inner membrane (Fig 2).


Figure 2. The Native E. coli EnvZ Receptor. The N- and C-termini of EnvZ are located in the cytoplasm, with two transmembrane domains separated by a periplasmic loop. The periplasmic loop senses membrane tension caused by osmotic stress. This tension is transmitted the cytoplasmic side of the protein and triggers auto-phosphorylation.

EnvZ sensor to detect microcystin

We want to replace the periplasmic domain of EnvZ with the PP1 protein (Fig 3), so that when microcystin binds to PP1 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 the GFP gene under control of the ompC promoter. This promoter is recognised and activated by phosphorylated OmpR and as a result, cells will turn green in the presence of microcystin, this acting as a microcystin detector.


Figure 3. Schematic representation of the engineered EnvZ microcystin detector. In the engineered construct PP1 replaces the periplasmic domain of EnvZ.

Progress

So far we have successfully cloned the 5’ and 3’ parts of envZ, replacing DNA encoding the periplasmic loop with that of the PP1 gene. Although this construct has been verified by sequencing, to date our attempts to express this hybrid protein have been unsuccessful. A possible reason for this is that the hybrid receptor is not correctly assembled. The periplasmic part of the receptor is translocated across the membrane by the Sec pathway, and we have already seen in our mop experiments that PP1 cannot be transported by Sec (possibly due to the presence of 13 cysteine residues in PP1 that may be aberrantly disulphide-bonded after translocation). It may be possible to overcome these limitations by re-engineering our hybrid EnvZ to interact with the Tat rather than the Sec pathway. The reporter under the control of OmpR has successfully been constructed, and confirmed to respond to OmpR by a change in expression of GFP.

Characterisation of our receptors

We ultimately want 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 have purchased microcystin to test our mop, and we can use this to bind and activate our receptors. We will then measure the amount of fluorescence by flow cytometry or microscopy. Then we can quantify the expression of GFP in relation to how much microcystin is presented to our cells. Using the number of receptors expressed in the membrane/spore for each cell we can calculate the effectiveness of our detector.