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

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     <div class="sec white" id="abstractview">
 
     <div class="sec white" id="abstractview">
−
         <div>
+
          
−
            <h1>Abstract View</h1>
+
−
        </div>
+
 
         <div>
 
         <div>
  
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−
                 <h2>Motivation</h2>
+
                 <h2>OVERVIEW</h2>
 
                 <p>
 
                 <p>
 
                     IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed
 
                     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.
 
                     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
 
                     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
+
                     are extremely complicated and invasive to perform,involving colonoscopy or biopsy, 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
 
                     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
+
                     box like system, the causes of IBD still remain unknown, preventing proper cure to be developped. However, it is thougth
 
                     that a disbalance in the gut microbiota, associated with some genetic factor migth be a possible cause
 
                     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
 
                     of IBD. The current main issue is thus investigation in order to confirm and to establish which microbiota
Line 66: Line 64:
 
<i>figure 1</i>
 
<i>figure 1</i>
 
     </p>       
 
     </p>       
−
                 <h3>Assumption</h3>
+
                 <h3>REQUIREMENT</h3>
 
                 <p>
 
                 <p>
 
                     In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present
 
                     In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present
Line 78: Line 76:
  
 
             <div class="sec white">
 
             <div class="sec white">
−
                 <h3>System</h3>
+
                 <h3>SYSTEM</h3>
 
                 <p>
 
                 <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
+
                     As stated before, current diagnosis tools in IBD involve invasive solution such as colonoscopy and biopsy. And because of the lack of information regarding IBD causes, and the lack of reliability when analysing samples in vitro, research stagnates. Therefore an adaptive learning tool for in vivo diagnostic and investigation was designed. Our system is composed of a logic AND gate, a switch and a second AND gate. The switch acts as a memory bit that can be flipped from 0 to 1 when it detects both inflammation and a specific microbiota disbalance through the AND gate. We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. Our first AND gate sensor is sensitive to both microbiota specific AHL, and inflammation marker NO. A later version allow us to sense lactate, as recent studies tends to demonstrate its role in heavy case of IBD in children. For minimal system disruption, we chose E.Coli which is native to gut microbiota. A future improvement would be to transfer this circuit into a Lactobacillus since they are also native and non pathogenic.
−
                    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>
 
                 </p>
 
             </div>
 
             </div>
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     <div class="sec light_grey" id="systemoverview">
 
     <div class="sec light_grey" id="systemoverview">
 
         <div>
 
         <div>
−
             <h1>System Overview</h1>
+
             <h1>SYSTEM OVERVIEW</h1>
 
         </div>
 
         </div>
 
     <div>
 
     <div>
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             <div class="sec light_grey">
 
             <div class="sec light_grey">
−
                 <h2>Inputs</h2>
+
 
 +
                 <h2>DESIGN MOTIVATION</h2>
 +
<p>One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible.We designed our circuit to be as modular as possible. As the interest of this genetic circuit mainly lays when multiplexing is possible, we designed it such that you can associate any candidate signal just by changing the promoter. Thus, constructing a simple library of <i>AHL</i>, you can create a library of E. Coli capable to sense a wide range of microbiota signals.
 +
Moreover, our model shows that the system is easily tunable playing with <i>NorR</i>,<i> Esar</i>, and <i>integrase</i> production and degradation rates to fit to the required range of detection, and thus would be the best solution for a modular multiplexing investigation tool.
 +
</p>
 +
                <h3>INPUTS SENSOR</h3>
 
                 <p>
 
                 <p>
−
                     We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor
+
                     We need a <i>NO</i> sensor and a <i>AHL</i> sensor. Later we will also need a Lactate sensor, as recent researches tend to asses that lactate is present in extremely high quantity in some heavy case of IBD in children. Our input sensor is composed of a PnorV promoter associated with esaboxes situated downstream the promoter and upstream the reporter gene. In an improved version of this sensor, the esabox (to which EsaR can bind and act as a roadblock , preventing gene transcription) will be put in different place around the PnorV promoter. The idea is to see if competitive binding can bring better result than traditional independent gene activation and inhibition. More over it is known that PnorV activation mechanism under <i>NO/NorR </i>binding involves DNA looping aroung the promoter. As a consequence, a low efficiency of <i>EsaR</i> road block behavior is expected as the looping could prevent it from binding to the esaboxes.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
  
 
             <div class="sec light_grey">
 
             <div class="sec light_grey">
−
                 <h3>Switch</h3>
+
                 <h3>SWITCH</h3>
−
                 <p> When both NO and AHL are present the hybrid promoter is activated and lead to Bxb1 invertase protein production.
+
                 <p> We currently developed three different possible switches, based on integrase kinetic, <i>CRISP/Cas9</i> and the recently discovered <i>CRISP/Cpf1</i> complex. When both <i>NO</i> and <i>AHL</i> are present the hybrid promoter is activated and lead to <i>intregrase</i> protein production. <i>Intregrase</i> is a protein that is capable of binding to a particular DNA sequence referred as <i>AttP</i> and <i>AttD</i>. After binding the DNA sequence is cut and inverted. The switch acts here as a memory bit that can be flipped  from 0 to 1 in an irreversible way. The flipped sequence contains a constitutive promoter associated with esaboxes, and thus negatively regulated by <i>EsaR</i>.  
−
                     This then activates our irreversible switch
+
                      
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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             <div class="sec light_grey">
 
             <div class="sec light_grey">
−
                 <h3>Reporter</h3>
+
                 <h3>REPORTER</h3>
−
                 <p> Once the switch is activated, and when AHL is present, GFP is produced.
+
                 <p> On each side of the flipped sequence are placed a <i>mNectarine</i> and a <i>GFP</i> gene respectively. Depending of the flipping state of the DNA sequence, the cell produces either <i>mNectarine</i> of <i>GFP</i>. As explained above this gene expression is regulated by ,<i>EsaR</i>. Thus once the DNA is switched, the cell produces <i>GFP</i> if <i>AHL</i> is present in the medium.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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     <div class="sec white" id="geneticcircuit">
 
     <div class="sec white" id="geneticcircuit">
 
         <div>
 
         <div>
−
             <h1>Genetic circuit</h1>
+
             <h1>GENETIC CIRCUIT</h1>
 
         </div>
 
         </div>
 
         <div>
 
         <div>
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             <div class="sec white">
 
             <div class="sec white">
−
                 <h2>NO sensor</h2>
+
                 <h2>NO SENSOR</h2>
 
                 <p>
 
                 <p>
−
                     We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under
+
                     We use here the PnorV promoter, specific to <i>NO</i> sensing. We also use a constitutively produced <i>NorR</i> protein. <i>NorR</i> exist under
 
                     a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those
 
                     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
+
                     three dimer assemble in a hexameric ring like structure. When <i>NO</i> is present in the medium, it binds to
 
                     the structure and activate the promoter.
 
                     the structure and activate the promoter.
 
                 </p>
 
                 </p>
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             <div class="sec white">
 
             <div class="sec white">
−
                 <h2>AHL sensor</h2>
+
                 <h2>AHL SENSOR</h2>
 
                 <p>
 
                 <p>
−
                     In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR
+
                     In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like <i>NorR</i>
−
                     it exists as a dimer inb the cell. The dimer form binds to the esaboxes forming a roadblock and unabling
+
                     it exists as a dimer in 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
+
                     gene transcription when <i>NO</i> only is present. When <i>AHL</i> enter the cell it binds to the <i>EsaR</i> dimer and free
 
                     the promoter, allowing transcription.
 
                     the promoter, allowing transcription.
 
                 </p>
 
                 </p>
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             <div class="sec white">
 
             <div class="sec white">
−
                 <h2>Lactate sensor</h2>
+
                 <h2>LACTATE SENSOR</h2>
 
                 <p>
 
                 <p>
−
                     Recent studies highlighted the fact that Lactate seems to be overpresent in some very heavy case of IBD, expecially in children.
+
                     Recent studies highlighted the fact that Lactate seems to be over-present in some very heavy case of IBD, especially in children.
−
                     Thus it is also intersting yto investigate the role of Lactate in IBD occurences. We uses here a modified
+
                     Thus it is also interesting to investigate the role of Lactate in IBD occurrences. We uses here a modified
−
                     version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR
+
                     version of the Plac promoter. two <i>LldR</i> binding sites O1 and O2 are situated upstream the promoter. <i>LldR</i>
−
                     and LldD (Lactate -> Pyruvate catalysist) are constitutively produced. in absence of Lacatte, LldR binds
+
                     and <i>LldD</i> (<i>Lactate</i> -> <i>Pyruvate</i> catalyst) are constitutively produced. in absence of <i>Lactate</i>, <i>LldR</i> binds
 
                     to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it 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.
+
                     to the <i>LldR</i> dimer and free the promoter. We introduced <i>LldD</i> to increase the threshold of Lactate sensing.
 
                 </p>
 
                 </p>
 
             </div>
 
             </div>
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                 <h2>AND GATE</h2>
 
                 <h2>AND GATE</h2>
 
                 <p>
 
                 <p>
−
                     The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed
+
                     The AND gate is the association of both <i>NO</i> sensor and <i>AHL</i> or Lactate sensor. It is constituted by a hybrid promoter composed
 
                     of the PnorV promoter and the downstream esaboxes.
 
                     of the PnorV promoter and the downstream esaboxes.
 
                 </p>
 
                 </p>
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             <div class="sec white">
 
             <div class="sec white">
−
                 <h2>Switch Module</h2>
+
                 <h2>SWITCH MODULE</h2>
 
                 <p>
 
                 <p>
−
                     AND gate activation triggers Bxb1 production. Bxb1 is an invertase protein. Its role is to inverse the DNA strand containing
+
                     AND gate activation triggers integrase production. Its role is to inverse the DNA strand containing
 
                     the GFP gene.
 
                     the GFP gene.
 
                 </p>
 
                 </p>
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             <div class="sec white">
 
             <div class="sec white">
−
                 <h2>Reporter Module</h2>
+
                 <h2>REPORTER MODULE</h2>
 
                 <p>
 
                 <p>
 
                     the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.
 
                     the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.

Revision as of 09:08, 16 October 2016

PAVLOV'S COLI

DESIGN

OVERVIEW

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 and invasive to perform,involving colonoscopy or biopsy, 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 to be developped. 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.

Figure 1: Schematic view of the model structure.

figure 1

REQUIREMENT

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

As stated before, current diagnosis tools in IBD involve invasive solution such as colonoscopy and biopsy. And because of the lack of information regarding IBD causes, and the lack of reliability when analysing samples in vitro, research stagnates. Therefore an adaptive learning tool for in vivo diagnostic and investigation was designed. Our system is composed of a logic AND gate, a switch and a second AND gate. The switch acts as a memory bit that can be flipped from 0 to 1 when it detects both inflammation and a specific microbiota disbalance through the AND gate. We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. Our first AND gate sensor is sensitive to both microbiota specific AHL, and inflammation marker NO. A later version allow us to sense lactate, as recent studies tends to demonstrate its role in heavy case of IBD in children. For minimal system disruption, we chose E.Coli which is native to gut microbiota. A future improvement would be to transfer this circuit into a Lactobacillus since they are also native and non pathogenic.

SYSTEM OVERVIEW

Figure 2: System Overview

figure 2

DESIGN MOTIVATION

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that are reliably reproducible.We designed our circuit to be as modular as possible. As the interest of this genetic circuit mainly lays when multiplexing is possible, we designed it such that you can associate any candidate signal just by changing the promoter. Thus, constructing a simple library of AHL, you can create a library of E. Coli capable to sense a wide range of microbiota signals. Moreover, our model shows that the system is easily tunable playing with NorR, Esar, and integrase production and degradation rates to fit to the required range of detection, and thus would be the best solution for a modular multiplexing investigation tool.

INPUTS SENSOR

We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor, as recent researches tend to asses that lactate is present in extremely high quantity in some heavy case of IBD in children. Our input sensor is composed of a PnorV promoter associated with esaboxes situated downstream the promoter and upstream the reporter gene. In an improved version of this sensor, the esabox (to which EsaR can bind and act as a roadblock , preventing gene transcription) will be put in different place around the PnorV promoter. The idea is to see if competitive binding can bring better result than traditional independent gene activation and inhibition. More over it is known that PnorV activation mechanism under NO/NorR binding involves DNA looping aroung the promoter. As a consequence, a low efficiency of EsaR road block behavior is expected as the looping could prevent it from binding to the esaboxes.

SWITCH

We currently developed three different possible switches, based on integrase kinetic, CRISP/Cas9 and the recently discovered CRISP/Cpf1 complex. When both NO and AHL are present the hybrid promoter is activated and lead to intregrase protein production. Intregrase is a protein that is capable of binding to a particular DNA sequence referred as AttP and AttD. After binding the DNA sequence is cut and inverted. The switch acts here as a memory bit that can be flipped from 0 to 1 in an irreversible way. The flipped sequence contains a constitutive promoter associated with esaboxes, and thus negatively regulated by EsaR.

REPORTER

On each side of the flipped sequence are placed a mNectarine and a GFP gene respectively. Depending of the flipping state of the DNA sequence, the cell produces either mNectarine of GFP. As explained above this gene expression is regulated by ,EsaR. Thus once the DNA is switched, the cell produces GFP if AHL is present in the medium.

GENETIC CIRCUIT

Figure 3: Genetic Circuit

figure 3

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 hexameric 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 in 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 over-present in some very heavy case of IBD, especially in children. Thus it is also interesting to investigate the role of Lactate in IBD occurrences. 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 catalyst) are constitutively produced. in absence of Lactate, LldR binds to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds to the 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 integrase production. 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.

Thanks to the sponsors that supported our project: