Difference between revisions of "Team:ETH Zurich/Design"

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−
  <div class="sec light_grey" id="abstractview">
+
    <div class="sec light_grey" id="abstractview">
 
         <div>
 
         <div>
 
             <h1>Abstract View</h1>
 
             <h1>Abstract View</h1>
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                     <img src="https://static.igem.org/mediawiki/2016/c/c2/T--ETH_Zurich--ElectricalSchematic.svg">
 
                     <img src="https://static.igem.org/mediawiki/2016/c/c2/T--ETH_Zurich--ElectricalSchematic.svg">
 
                 </a>
 
                 </a>
−
                 <p><b>Figure 1:</b> System Overview</p>
+
                 <p><b>Figure 1:</b> Abstract View</p>
 
             </div>
 
             </div>
  
−
        <div class="sec white">
+
            <div class="sec white">
−
            <h2>Motivation</h2>
+
                <h2>Motivation</h2>
−
            <p>
+
                <p>
−
                In the absence of NO, NorR is produced constitutively and binds repressively to the PnorV promoter, preventing gene transcription.
+
                    IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed
−
                When NO is present in the medium, it binds cooperatively to the hexameric form of NorR,and activate the promoter.
+
                    from Ulceric colitis and Crohn disease each year. In Europe about 1.4 milliom persons are concerned.
−
            </p>
+
                    In the United state The number of infected poeple reach 3 millions. Moreover in vivo diagnostic and test
−
        </div>
+
                    are extremely complicated to perform, while in vitro experiment are not reliable enough, because the
 +
                    intestine environement cannot be properly mimicated outside the human body. As the gut remain a black
 +
                    box like system, the causes of IBD still remain unknown, preventing proper cure. However, it is thougth
 +
                    that a disbalance in the gut microbiota, associated with some genetic factor migth be a possible cause
 +
                    of IBD. The current main issue is thus investigation in order to confirm and to establish which microbiota
 +
                    extending or shrinking can be associated with iBD.
 +
                </p>
 +
            </div>
  
−
        <div class="sec white">
+
            <div class="sec white">
−
            <h3>Assumption</h3>
+
                <h3>Assumption</h3>
−
            <p> We considered here that the binding of NO to NorR and PnorV_{i} does not affect the other species binding. Thus
+
                <p>
−
                the reactions \begin{align*} NorR+NO&amp;\rightleftharpoons NorR_{NO}\\ \end{align*} and \begin{align*} PnorV_{NorR}+NO&amp;\rightleftharpoons
+
                    In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present
−
                NorR_{NO}\\ \end{align*} have the same reaction rate. Under those assumption, the system of equation can
+
                    in the gut when there are lesion and inflammation. We also need a proper microbiota marker. Each microbiota
−
                thus be simplified as follows:
+
                    naturally produces specific AHL. We thus chose AHL as a microbiota specific marker. Finally, we also
−
            </p>
+
                    need a reporter. We chose Mnectarine a redish fluorescent protein, that will be expressed in case of
−
        </div>
+
                    absence of associated inflammation with the microbiota, and GFP expressed in case a match between one
 +
                    specific microbiota and inflammation is found.
 +
                </p>
 +
            </div>
  
 +
            <div class="sec white">
 +
                <h3>System</h3>
 +
                <p>
 +
                    What dkind of circuit do we need we need to achieve our goal ? We need to associate two signals. A AND Gate seems to be the
 +
                    most appropriate circuit to be implemented in order to do that. However, as said previously, the gut
 +
                    behave as a black box. We need our bacteria to enter the gut, and we need to be able to know with which
 +
                    chemicals they interact once we separate them from the faeces. A simple AND Gate is here nott enough
 +
                    because any GFP fluorescent would have disappear in the timelaps between signal apparition and bacteria
 +
                    harvesting. We thus need our bacteria to 'remember' what happenned. To achieve this, we use a irreversible
 +
                    switch activated when both NO and AHL where present simultaneously. Then Un der the switch activation,
 +
                    GFP can be expressed. Later simply exposing the bacteria to AHL make them produce GFP, allowing us to
 +
                    know which microbiota was overactive when inflammation happened.
 +
                </p>
 +
            </div>
  
  
 +
        </div>
 
     </div>
 
     </div>
−
</div>
 
  
  
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                     <img src="https://static.igem.org/mediawiki/2016/d/dd/T--ETH_Zurich--ModularView.svg">
 
                     <img src="https://static.igem.org/mediawiki/2016/d/dd/T--ETH_Zurich--ModularView.svg">
 
                 </a>
 
                 </a>
−
                 <p><b>Figure 1:</b> System Overview</p>
+
                 <p><b>Figure 2:</b> System Overview</p>
 
             </div>
 
             </div>
  
  
−
        <div class="sec white">
+
            <div class="sec white">
−
            <h2>Motivation</h2>
+
                <h2>Inputs</h2>
−
            <p>
+
                <p>
−
                In the absence of NO, NorR is produced constitutively and binds repressively to the PnorV promoter, preventing gene transcription.
+
                    We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor
−
                 When NO is present in the medium, it binds cooperatively to the hexameric form of NorR,and activate the promoter.
+
                 </p>
−
            </p>
+
            </div>
−
        </div>
+
  
−
        <div class="sec white">
+
            <div class="sec white">
−
            <h3>Assumption</h3>
+
                <h3>Switch</h3>
−
            <p> We considered here that the binding of NO to NorR and PnorV_{i} does not affect the other species binding. Thus
+
                <p> When both NO and AHL are present the hybrid promoter is activated and lead to Bxb1 invertase protein production.
−
                the reactions \begin{align*} NorR+NO&amp;\rightleftharpoons NorR_{NO}\\ \end{align*} and \begin{align*} PnorV_{NorR}+NO&amp;\rightleftharpoons
+
                    This then activates our irreversible switch
−
                 NorR_{NO}\\ \end{align*} have the same reaction rate. Under those assumption, the system of equation can
+
                 </p>
−
                thus be simplified as follows:
+
            </div>
−
            </p>
+
−
        </div>
+
  
  
  
 +
            <div class="sec white">
 +
                <h3>Reporter</h3>
 +
                <p> Once the switch is activated, and when AHL is present, GFP is produced.
 +
                </p>
 +
            </div>
 +
 +
 +
        </div>
 
     </div>
 
     </div>
−
</div>
 
  
 
     <div class="sec light_grey" id="geneticcircuit">
 
     <div class="sec light_grey" id="geneticcircuit">
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                     <img src="https://static.igem.org/mediawiki/2016/e/e5/T--ETH_Zurich--GeneticCircuit.svg">
 
                     <img src="https://static.igem.org/mediawiki/2016/e/e5/T--ETH_Zurich--GeneticCircuit.svg">
 
                 </a>
 
                 </a>
−
                 <p><b>Figure 1:</b> System Overview</p>
+
                 <p><b>Figure 3:</b> Genetic Circuit</p>
 
             </div>
 
             </div>
  
Line 104: Line 132:
 
                 <h2>NO sensor</h2>
 
                 <h2>NO sensor</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under
−
                     promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
                    a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
                     three dimer assemble in a hexamric ring like structure. When NO is present in the medium, it binds to
−
                    EsaBox can be added, in order to tune the sensor sensitivity.
+
                    the structure and activate the promoter.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
Line 114: Line 142:
 
                 <h2>AHL sensor</h2>
 
                 <h2>AHL sensor</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR
−
                     promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
                    it exists as a dimer inb the cell. The dimer form binds to the esaboxes forming a roadblock and unabling
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
                     gene transcription when NO only is present. When AHL enter the cell it binds to the EsaR dimer and free
−
                    EsaBox can be added, in order to tune the sensor sensitivity.
+
                    the promoter, allowing transcription.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
Line 124: Line 152:
 
                 <h2>Lactate sensor</h2>
 
                 <h2>Lactate sensor</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     Recent studies highlighted the fact that Lactate seems to be overpresent in some very heavy case of IBD, expecially in children.
−
                     promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
                    Thus it is also intersting yto investigate the role of Lactate in IBD occurences. We uses here a modified
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
                    version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR
−
                    EsaBox can be added, in order to tune the sensor sensitivity.
+
                     and LldD (Lactate -> Pyruvate catalysist) are constitutively produced. in absence of Lacatte, LldR binds
 +
                    to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds
 +
                    to tyhe LldR dimer and free the promoter. We introduced LldD to increase the threshold of Lactate sensing.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
Line 134: Line 164:
 
                 <h2>AND GATE</h2>
 
                 <h2>AND GATE</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed
−
                     promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
                     of the PnorV promoter and the downstream esaboxes.
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
−
                    EsaBox can be added, in order to tune the sensor sensitivity.
+
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
Line 144: Line 172:
 
                 <h2>Switch Module</h2>
 
                 <h2>Switch Module</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     AND gate activation triggers Bxb1 production. Bxb1 is an invertase protein. Its role is to inverse the DNA strand containing
−
                    promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
                     the GFP gene.
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
−
                     EsaBox can be added, in order to tune the sensor sensitivity.
+
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
Line 154: Line 180:
 
                 <h2>Reporter Module</h2>
 
                 <h2>Reporter Module</h2>
 
                 <p>
 
                 <p>
−
                     In the absence of AHL, EsaR is constitutively produced, dimerizes and bind as a dimer to the esaBox situated downstream the
+
                     the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.
−
                    promoter, preventing transcription as a roadblock. When a higher than normal amount of AHL is present
+
−
                    in the gut, it binds to the EsaR dimer, and free the promoter, allowing transcription. Later on, several
+
−
                    EsaBox can be added, in order to tune the sensor sensitivity.
+
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
  
−
           
 
  
−
            <p>
+
 
−
                We assume a very fast dimerization of EsaR
+
 
−
            </p>
+
 
         </div>
 
         </div>
 
     </div>
 
     </div>

Revision as of 09:51, 13 September 2016

PAVLOV'S COLI

Abstract View

Figure 1: Abstract View

Motivation

IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed from Ulceric colitis and Crohn disease each year. In Europe about 1.4 milliom persons are concerned. In the United state The number of infected poeple reach 3 millions. Moreover in vivo diagnostic and test are extremely complicated to perform, while in vitro experiment are not reliable enough, because the intestine environement cannot be properly mimicated outside the human body. As the gut remain a black box like system, the causes of IBD still remain unknown, preventing proper cure. However, it is thougth that a disbalance in the gut microbiota, associated with some genetic factor migth be a possible cause of IBD. The current main issue is thus investigation in order to confirm and to establish which microbiota extending or shrinking can be associated with iBD.

Assumption

In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present in the gut when there are lesion and inflammation. We also need a proper microbiota marker. Each microbiota naturally produces specific AHL. We thus chose AHL as a microbiota specific marker. Finally, we also need a reporter. We chose Mnectarine a redish fluorescent protein, that will be expressed in case of absence of associated inflammation with the microbiota, and GFP expressed in case a match between one specific microbiota and inflammation is found.

System

What dkind of circuit do we need we need to achieve our goal ? We need to associate two signals. A AND Gate seems to be the most appropriate circuit to be implemented in order to do that. However, as said previously, the gut behave as a black box. We need our bacteria to enter the gut, and we need to be able to know with which chemicals they interact once we separate them from the faeces. A simple AND Gate is here nott enough because any GFP fluorescent would have disappear in the timelaps between signal apparition and bacteria harvesting. We thus need our bacteria to 'remember' what happenned. To achieve this, we use a irreversible switch activated when both NO and AHL where present simultaneously. Then Un der the switch activation, GFP can be expressed. Later simply exposing the bacteria to AHL make them produce GFP, allowing us to know which microbiota was overactive when inflammation happened.

System Overview

Figure 2: System Overview

Inputs

We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor

Switch

When both NO and AHL are present the hybrid promoter is activated and lead to Bxb1 invertase protein production. This then activates our irreversible switch

Reporter

Once the switch is activated, and when AHL is present, GFP is produced.

Genetic circuit

Figure 3: Genetic Circuit

NO sensor

We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those three dimer assemble in a hexamric ring like structure. When NO is present in the medium, it binds to the structure and activate the promoter.

AHL sensor

In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR it exists as a dimer inb the cell. The dimer form binds to the esaboxes forming a roadblock and unabling gene transcription when NO only is present. When AHL enter the cell it binds to the EsaR dimer and free the promoter, allowing transcription.

Lactate sensor

Recent studies highlighted the fact that Lactate seems to be overpresent in some very heavy case of IBD, expecially in children. Thus it is also intersting yto investigate the role of Lactate in IBD occurences. We uses here a modified version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR and LldD (Lactate -> Pyruvate catalysist) are constitutively produced. in absence of Lacatte, LldR binds to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds to tyhe LldR dimer and free the promoter. We introduced LldD to increase the threshold of Lactate sensing.

AND GATE

The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed of the PnorV promoter and the downstream esaboxes.

Switch Module

AND gate activation triggers Bxb1 production. Bxb1 is an invertase protein. Its role is to inverse the DNA strand containing the GFP gene.

Reporter Module

the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.