Team:Dundee/Project/Detector

From 2013.igem.org

(Difference between revisions)
(Created page with "<html> <html lang="en"> <head> <meta charset="utf-8"> <title>iGEM Dundee 2013 · Toxi-Mop</title> <meta name="viewport" content="width=device-width, initial-s...")
 
(10 intermediate revisions not shown)
Line 1: Line 1:
 +
{{:Team:Dundee/Templates/Navigationbar}}
 +
<html>
<html>
<html lang="en">
<html lang="en">
-
  <head>
 
-
    <meta charset="utf-8">
 
-
    <title>iGEM Dundee 2013 &middot; Toxi-Mop</title>
 
-
    <meta name="viewport" content="width=device-width, initial-scale=1.0">
 
-
    <meta name="description" content="">
 
-
    <meta name="author" content="">
 
-
    <!-- CSS -->
+
      <!-- Begin page content -->
-
    <link href=' http://fonts.googleapis.com/css?family=Open+Sans' rel='stylesheet' type='text/css'>
+
      <div class="container">
-
    <link href="http://www.kyleharrison.co.uk/igem/assets/css/bootstrap.css" rel="stylesheet">
+
-
    <link href="http://www.kyleharrison.co.uk/igem/assets/css/style.css" rel="stylesheet">
+
-
    <!--
+
      <!-- Title -->
-
    <link href="http://www.kyleharrison.co.uk/igem/assets/css/bootstrap-responsive.css" rel="stylesheet">
+
      <div class="page-header">
-
-->
+
          <h2><b>The Detector</b> </h2>
-
    <!-- HTML5 shim, for IE6-8 support of HTML5 elements -->
+
        </div>
-
    <!--[if lt IE 9]>
+
      <!-- Title End -->
-
      <script src="http://www.kyleharrison.co.uk/igem/assets/js/html5shiv.js"></script>
+
-
    <![endif]-->
+
-
    <!-- Fav and touch icons -->
+
<!-- Detector 2 -->
-
    <link rel="apple-touch-icon-precomposed" sizes="144x144" href="../assets/ico/apple-touch-icon-144-precomposed.png">
+
-
    <link rel="apple-touch-icon-precomposed" sizes="114x114" href="../assets/ico/apple-touch-icon-114-precomposed.png">
+
-
      <link rel="apple-touch-icon-precomposed" sizes="72x72" href="../assets/ico/apple-touch-icon-72-precomposed.png">
+
-
                    <link rel="apple-touch-icon-precomposed" href="../assets/ico/apple-touch-icon-57-precomposed.png">
+
-
                                  <link rel="shortcut icon" href="../assets/ico/favicon.png">
+
-
  </head>
+
-
  <body>
+
      <div class="row" style="text-align:justify;margin-top:-20px;">
 +
        <div class="span12">
 +
          <h2>The detection systems</h2>
 +
We designed two systems to detect microcystin, one in each of our chassis organisms.<br><br>
-
    <!-- Part 1: Wrap all page content here -->
+
<h2>Engineering the <i>B. subtilis</i> PrkC receptor to respond to microcystin</h2>
-
    <div id="wrap">
+
<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>  
 +
<h2>Why sense cell wall peptides? How does this indicate that conditions are permissive for growth?</h2>
 +
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>
-
      <!-- Fixed navbar -->
+
<h2>The PrkC receptor</h2>
-
      <div class="navbar navbar-fixed-top">
+
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>
-
        <div class="navbar-inner">
+
<h2>But how can we use the PrkC receptor to detect microcystin?</h2>
 +
We hope to detect microcystin by replacing the 3 ligand binding domains with three copies of PP1 (Fig 1B).<br><br>
-
          <div class="container">
 
-
            <button type="button" class="btn btn-navbar" data-toggle="collapse" data-target=".nav-collapse">
 
-
              <span class="icon-bar"></span>
 
-
              <span class="icon-bar"></span>
 
-
              <span class="icon-bar"></span>
 
-
            </button>
 
-
            <a class="brand" href="/Team:Dundee">Dundee iGEM 2013</a>
 
-
            <div class="nav-collapse collapse">
 
-
              <ul class="nav">
 
-
                <li class="active"><a href="/Team:Dundee">Home</a></li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Team <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="/Team:Dundee/Team">Meet the Team</a></li>
 
-
                    <li><a href="/Team:Dundee/Team/Gallery">Gallery</a></li>
 
-
                    <li><a href="https://igem.org/Team.cgi?id=1012">Team Information</a></li>
 
-
                    <li><a href="/Team:Dundee/Team/Awknowledgements">Acknowledgements</a></li>
 
-
                    <li><a href="/Team:Dundee/Team/Attributions">Attributions</a></li>
 
-
                    <li><a href="#">Contact</a></li>
 
-
                  </ul>
 
-
                </li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Project <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="./Project/Overview.html">Project Overview</a></li>
 
-
                    <li><a href="#">Notebook</a></li>
 
-
                    <li class="divider"></li>
 
-
                    <li class="nav-header">Lab</li>
 
-
                    <li><a href="#">Lab Overview</a></li>
 
-
                    <li><a href="#">Detector</a></li>
 
-
                    <li><a href="#">Sensor</a></li>
 
-
                    <li class="divider"></li>
 
-
                    <li class="nav-header">Modelling </li>
 
-
                    <li><a href="./Project/MathOverview.html">Modelling Overview</a></li>
 
-
                    <li><a href="./Project/MathOverview.html">Theory</a></li>
 
-
                    <li class="divider"></li>
 
-
                    <li class="nav-header">Software</li>
 
-
                  <li><a href="#">Software Overview</a></li>
 
-
                  <li><a href="#">Mop-toppus</a></li>
 
-
                  <li><a href="#">Toxi-Tweet</a></li>
 
-
                  </ul>
 
-
                </li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Parts <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="#">Our Biobricks</a></li>
 
-
                    <li><a href="#">Improvements</a></li>
 
-
                  </ul>
 
-
                </li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Safety <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="#">General Safety</a></li>
 
-
                    <li><a href="#">Safety in the Lab</a></li>
 
-
                    <li><a href="#">Public Safety and Awareness</a></li>
 
-
                    <li><a href="#">Enviromental Safety</a></li>
 
-
                  </ul>
 
-
                </li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Human Practice <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="#">Overview</a></li>
 
-
                    <li><a href="#">Collaboration</a></li>
 
-
                    <li><a href="#">Outreach</a></li>
 
-
                    <li class="divider"></li>
 
-
                    <li class="nav-header">Media</li>
 
-
                    <li><a href="http://www.youtube.com/channel/UCvHOQ9Y1PqKInj6iCwLqTJw/feed?view_as=public">Youtube Channel</a></li>
 
-
                    <li><a href="#">Graphic Novel</a></li>
 
-
                    <li><a href="http://www.flickr.com/photos/97927329@N05/">Flickr</a></li>
 
-
                    <li><a href="#">Video Game</a></li>
 
-
                    <li class="divider"></li>
 
-
                    <li class="nav-header">Social Media</li>
 
-
                    <li><a href="https://www.facebook.com/DundeeiGem2013">Facebook</a></li>
 
-
                    <li><a href="https://twitter.com/DundeeiGEMTeam">Twitter</a></li>
 
-
                    <li><a href="https://plus.google.com/u/0/116223511035478208262/posts?hl=en_US">Google+</a></li>
 
-
                   
 
-
                  </ul>
 
-
                </li>
 
-
                <li class="dropdown">
 
-
                  <a href="#" class="dropdown-toggle" data-toggle="dropdown">Sponsors <b class="caret"></b></a>
 
-
                  <ul class="dropdown-menu">
 
-
                    <li><a href="#">Our Sponsors</a></li>
 
-
                    <li><a href="#">Sponsorship Levels</a></li>
 
-
                  </ul>
 
-
                </li>
 
-
              </ul>
 
-
            </div><!--/.nav-collapse -->
 
-
          </div>
 
-
        </div>
 
-
      </div>
 
 +
<center><img src="http://farm8.staticflickr.com/7292/10034973665_c91f4f9ea7_o.jpg"><br></center>
 +
<strong>Figure 1. : The <i>B. subtilis</i> PrkC Receptor (A) and the engineered receptor designed to respond to microcystin (B). </strong><br><br>
 +
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>
 +
<h2>Progress so far</h2>
 +
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>
-
      <!-- Begin page content -->
+
<h2>Engineering the <i>E. coli</i> EnvZ sensor kinase to respond to microcystin</h2>
-
      <div class="container">
+
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>
-
      <!-- Title -->
+
<h2>EnvZ sensor kinase</h2>
-
      <div class="page-header">
+
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>
-
          <h2><b>The Detector</b> </h2>
+
-
        </div>
+
-
      <!-- Title End -->
+
-
      <div class="row" style="text-align:justify;margin-top:-20px;">
 
-
        <div class="span12">
 
-
          <h2>Aims:</h2>
 
-
          <p>Using mathematical tools to allow us to predict the limiting factors in the production of PP1 and its mopping applications. Working alongside the biologists to produce models which are relevant and can predict what is expected to happen during the synthetic engineering of the mop and detection bacteria.</p>
 
-
        </div>
 
-
        <div class="span6">
 
-
        </div>
 
-
      </div><!-- Row End -->
 
-
            <div class="row" style="text-align:justify">
+
<center><img src="http://farm3.staticflickr.com/2859/10035077703_80c60c236e_o.jpg"></center><br>
-
        <div class="span6">
+
<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>Detection Comparison:</h2>
+
-
          <p> The current method for detecting toxic levels of microcystin is to take a sample of water from different regions of the site being investigated and then to carry out high performance liquid chromatography (HPLC). This process currently takes approximately 24 hours, we hope to reduce this to a more suitable 1 hour.</p><br>
+
-
          <p>Assuming the cyanobacteria undergo binary fission and grow unbounded we were able to determine how the problem increases over 24 hours in comparison to 1 hour detection.
+
-
          where MC(t) is the number of microcystin at time t b0 is the initial number of algae</p><br>
+
-
          <p>The ratio for time t=24:1 is 8.4million:1. To put this into perspective this is the same as the height of the empire state building compared with the length of 7 E.coli bacterium. This model therefore emphasises that the 1 hour detection period is much more efficient and worth pursuing.</p>
+
-
        </div>
+
-
          <div class="span6" style="margin-top:60px;">
+
<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>
-
            <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Equation">
+
-
          </div><br>
+
-
      </div><!-- Row End -->
+
<center><img src="http://farm8.staticflickr.com/7415/10035089443_977abb0772_o.jpg"></center><br>
 +
<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>
 +
<h2>Progress</h2>
-
        <div class="row" style="text-align:justify">
+
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.
-
        <div class="span6">
+
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>
 +
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>
-
        <h2>Geometric Packing:</h2>
+
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.
-
        <p>We considered different limiting factors of our mop bacteria.  The factor discussed in this section is the maximum number of PP1 which can fit either on the surface of B.subtilis, or in the periplasm of E.coli.  We considered the volumes of the bacteria and PP1 and used a cube approximation that took into account volume which was wasted, in packing, by the spherical shape of the protein. For this model we assumed there were no other surface proteins and protein production was not limited by any factors.</p><br>
+
-
       
+
-
        <p>Calculations show the maximum number of PP1 which can fit on the surface of B.subtilis is between 60 000 -70 000. From the average we can calculate that the number of bacterial mops required to clean a toxic level of microcystin in a litre of water is 1.40x1010.</p><br>
+
-
       
+
-
        <p>In E.coli, PP1 which would bind microcystin is free-flowing in the periplasm. The volume of the periplasm is much greater than the surface of B.subtilis. Therefore E.coli has the capacitive potential to be a more efficient mop. The maximum number of PP1 which can be packed into the periplasm is between 150 000 -200 000. Consequently, less bacterial mops are required to clean the same level of microcystin: 0.52x1010.</p><br>
+
-
       
+
-
        <p>When we have accurate numbers from the biology team on how many PP1 are attached to the surface or in the periplasm for B.subtilis and E.coli respectively, we can compare these numbers and compute the efficiency of our PP1 expressing bacteria.</p><br>
+
-
        <h2>Transcription, Translation </h2><br>
 
-
        <p>An Ordinary Differential Equation (ODE) uses a function f(t) to describe how the output changes as a result of changing the input dx(t)/dt. For example how PP1 concentration changes with time in a single cell. In order to model transcription and translation of PP1 we used a system of ODEs , which is more than one ODE where the outputs are coupled.</p><br>
 
-
        <p>We used law of mass action to obtain a system of ODEs to describe the production of mRNA to PP1. mRNA and PP1 are coupled in the sense we need mRNA before we can produce any PP1. Also, the mRNA is not used up. We also took into consideration the degradation rates of mRNA and PP1 which are denoted as .</p><br>
 
-
        <ul>
 
-
        <li>k1 – rate mRNA production - 4.98x10-9</li>
 
-
        <li>kd1 – rate mRNA degradation – 1x10-2</li>
 
-
        <li>k2 – rate PP1 production – 4x10-2</li>
 
-
        <li>kd2 – rate PP1 degradation – 4x10-4</li>
 
-
        </ul>
 
-
        </div>
 
-
          <div class="span6" style="margin-top:60px;">
 
-
            <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Figure 1">
 
-
          </div><br>
 
-
 
-
          <div class="span6" >
 
-
        <br> <p><i><b>Figure 1.</b> How mRNA and PP1 are produced over 20 minute cell division time. Note scaling on PP1 compared to mRNA.</i></p><br>
 
-
          </div>
 
-
 
-
          <div class="span6">
 
-
            <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Figure 2">
 
-
          </div><br><br>
 
-
 
-
          <div class="span6">
 
-
          <p><br><i>Figure 2. A steady state is when the quantities describing a system are independent of time – they reach an equilibrium i.e dx/dt = 0. The steady state for (mRNA, PP1) is (0.04, 0.04) corresponding to a non-dimensionalised system. This plot demonstrates that during a 20 minute cell division period mRNA reaches the steady state but PP1 does not.</i></p><br>
 
         </div>
         </div>
-
 
+
          
-
          <div class="span6">
+
-
            <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Figure 3">
+
-
          </div><br>
+
-
 
+
-
        <div class="span6">
+
-
         <br><p><i>Figure 3. This plot shows that given a time longer than cell division time both the mRNA and PP1 eventually reach their steady states.</i></p><br>
+
-
        </div>
+
-
 
+
       </div><!-- Row End -->
       </div><!-- Row End -->
-
 
+
   

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.