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

 
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        <li class="outline_item"><a href="#abstractview">Abstract View</a></li>
 
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        <li class="outline_item"><a href="#systemoverview">System Overview</a></li>
 
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        <li class="outline_item"><a href="#geneticcircuit">Genetic Circuit</a></li>
 
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<div class = "sec page_title">
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<div class="sec page_title">
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<div>
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    <h1>DESIGN</h1>
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<h1>DESIGN</h1>
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</div>
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</div>
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</div>
  
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<div class="sec white">
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    <div class="sec white" id="abstractview">
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<div>
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        <div>
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<h2>OVERVIEW</h2>
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            <h1>Abstract View</h1>
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<!--                  IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed
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                <h2>Motivation</h2>
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                <p>
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                    IBD has recently becoma a major issue in devellopped European country. In the past few years, around 100 000 are newly diagnosed
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                     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
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                     are extremely complicated to perform, while in vitro experiment are not reliable enough, because the
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                     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
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                     box like system, the causes of IBD still remain unknown, preventing proper cure. However, it is thougth
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                     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
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                     extending or shrinking can be associated with iBD.
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                     extending or shrinking can be associated with iBD.-->
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                </p>
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<p>Inflammatory bowel disease (IBD) is an insufficiently understood chronic disease, whose increasing incidence, lack of
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            </div>
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proper treatment and diagnostic methods make it an urgent problem to solve<sup><a href="#references">[1]</a></sup>. The current methods
 +
for diagnosing IBD mainly rely on endoscopies, which are invasive and do not allow to properly characterize the microbiome of patients, believed to be implicated in pathogenesis.</p>
  
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<p>We designed an alternative solution for <i>in vivo</i> diagnosis, using hydrogel-encapsulated Pavlov's coli. In order
 +
for Pavlov's coli to efficiently gather information on important aspects of IBD, such as inflammation and microbiome
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composition, we designed our system such that it consists of:</p>
  
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<ol>
 +
<li>A biological AND gate that allows our system to associate known IBD markers present in the gut.</li>
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<li>A switch acting as memory, which is irreversibly flipped from "OFF" to "ON" if both markers are simultaneously detected
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by the AND gate. This switching mechanism renders a reporter (GFP, see later) sensitive to one of the two biomarkers
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previously encountered by our logic AND gate.</li>
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<li>Information sensed by the AND gate is stored (remembered) by the reporter via the switching mechanism and can be read-out
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later in a laboratory. This conditional activation of our reporter justifies the name of our diagnostic tool - Pavlov's
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coli.
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</li>
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</ol>
  
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              <div class="image_box full_size">
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<p>
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                    <a href="https://2016.igem.org/File:T--ETH_Zurich--model_structure.svg">
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The encapsulation should allow for diffusion of molecules, while preventing our bacteria from colonizing the gut, therefore
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                        <img src="https://static.igem.org/mediawiki/2016/4/44/T--ETH_Zurich--model_structure.svg">
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providing a non-invasive tool, which does not disrupt the microbiome.
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                    </a>
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</p>
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                    <p><b>Figure 1:</b> Schematic view of the model structure.</p>
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                </div>
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<div class="image_box full_size" id="fig1">
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<a href="https://2016.igem.org/File:T--ETH_Zurich--model_structure.png">
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<img src="https://static.igem.org/mediawiki/2016/1/15/T--ETH_Zurich--model_structure.png">
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</a>
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<p><b>Fig. 1:</b> Structure of Pavlov's coli's genetic circuit. (1) The sensor module comprises an AND gate which associates
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an inflammation marker with a candidate marker, representative of microbiome composition. (2) The switch module consists
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of an irreversible genetic switch. (3) The reporter module can be used to retrieve information about the biomarkers
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sensed by our AND gate.</p>
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</div>
  
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<p>
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                <a href="https://2016.igem.org/File:T--ETH_Zurich--ElectricalSchematic">
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An additional feature of our system is that it allows for multiplexing, meaning processing multiple signals through one channel:
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                    <img src="https://static.igem.org/mediawiki/2016/c/c2/T--ETH_Zurich--ElectricalSchematic.svg">
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in our system, the reporter module (3) is activated by one of the two markers also required to activate the sensor module,
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                </a>
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i.e. the AND gate (1) (see <a href="#fig1">Fig. 1</a>). The architecture of our system has the following implication:
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                <p>Figure 1: Abstract View</p>
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a suite of bacterial populations, each responsive to different IBD-relevant candidate markers (e.g. different AHL molecules,
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            </div>
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lactate, etc.) as well as to an inflammation marker (e.g. NO), can be engineered, complexed together and administered
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-->
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to a patient; upon recovery, re-exposure of faecal samples to a previously encountered biomarker can be read out through
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      <p>
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our reporter module (see <a href="#fig2">Fig. 2</a>).
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<i>figure 1</i>
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</p>
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    </p>     
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                <h3>Assumption</h3>
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                <p>
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                    In order to carry this investigation, we need a factor specific to inflammation. Here, we choose Nitric Oxide, which is present
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                    in the gut when there are lesion and inflammation. We also need a proper microbiota marker. Each microbiota
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                    naturally produces specific AHL. We thus chose AHL as a microbiota specific marker. Finally, we also
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                    need a reporter. We chose Mnectarine a redish fluorescent protein, that will be expressed in case of
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                    absence of associated inflammation with the microbiota, and GFP expressed in case a match between one
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                    specific microbiota and inflammation is found.
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                </p>
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            <div class="sec white">
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<div class="image_box full_size" id="fig2">
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                <h3>System</h3>
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<a href="https://2016.igem.org/File:T--ETH_Zurich--wikihome.png">
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                <p>
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<img src="https://static.igem.org/mediawiki/2016/9/9a/T--ETH_Zurich--wikihome.png">
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                    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
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</a>
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                    most appropriate circuit to be implemented in order to do that. However, as said previously, the gut
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<p><b>Fig. 2:</b>Mode of action of Pavlov's coli. Step 1: After administration of our bacteria, biological markers can be
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                    behave as a black box. We need our bacteria to enter the gut, and we need to be able to know with which
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sensed in the gut through a logical AND gate; the event is memorized through a switching mechanism. Step 2: Following
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                    chemicals they interact once we separate them from the faeces. A simple AND Gate is here nott enough
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excretion and retrieval of our bacteria, the memory can be read-out by exposing the samples containing our bacteria
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                    because any GFP fluorescent would have disappear in the timelaps between signal apparition and bacteria
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to a host of IBD-relevant candidate markers. </p>
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                    harvesting. We thus need our bacteria to 'remember' what happenned. To achieve this, we use a irreversible
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</div>
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                    switch activated when both NO and AHL where present simultaneously. Then Un der the switch activation,
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                    GFP can be expressed. Later simply exposing the bacteria to AHL make them produce GFP, allowing us to
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                    know which microbiota was overactive when inflammation happened.
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                </p>
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            </div>
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<p>
 +
Therefore, Pavlov's coli holds promise as a non-invasive, modular, multipleable, <i>in vivo</i> diagnostic tool for IBD
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investigation.
 +
</p>
 +
</div>
 +
<div>
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<h3>Sensing in the gut</h3>
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<p>One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that
 +
remain reliable. We designed our circuit as interchangeable, easily tunable parts, each of which is backed by a computer
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model and experimental data.</p>
  
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        </div>
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<p>
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    </div>
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IBD is normally accompanied by severe inflammation of the intestine. Several sources in the literature report on a sharp
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increase in Nitric Oxide (NO) levels in inflamed areas. Based on this, we chose to incorporate a sensor for NO as part
 +
of a logic AND gate in our engineered bacteria<sup><a href="#references">[1]</a></sup>.
 +
</p>
  
 +
<p>
 +
Along with the presence of NO, Pavlov's coli's logic gate requires a second input in order to activate an irreversible switch
 +
(see <a href="#fig1">Fig. 1</a>; also, see section below). As the second input, we chose a marker which can provide
 +
information on microbiome composition: AHL. A number of AHLs have been found to be informative as to specific functions
 +
(e.g. biofilm formation) and microbiome composition<sup><a href="#references">[2]</a></sup>.
 +
</p>
  
 +
<p>
 +
In an alternative design, we chose to replace the AHL sensor with lactate sensor. In an interview with an expert on IBD,
 +
Prof. Lacroix, we discussed the potential of bacterial metabolites as an
 +
alternative microbiome marker. One such a candidate turned out to be lactate, which is produced and metabolized by different
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bacteria in the gut. Although it is not as informative as AHL to the end of microbiome characterization, there is a strong
 +
interest in developing methods for lactate detection in the gut. Due to its nature as an intermediate metabolite, it
 +
is currently not possible to measure lactate concentrations in fecal samples of patients, adding appeal to the development
 +
of Pavlov's coli.
 +
</p>
 +
</div>
 +
</div>
  
 +
<div class="sec light_grey" id="systemoverview">
 +
<div>
 +
<h2>DETAILED IMPLEMENTATION</h2>
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<p>
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Our system is composed of a logic AND gate, a switch and a reporter, behaving together as an associative learning circuit.
 +
</p>
  
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    <div class="sec light_grey" id="systemoverview">
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<p>
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        <div>
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The AND gate is composed of the P<sub>norV</sub> promoter and an operator. The promoter can be activated by NorR upon
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            <h1>System Overview</h1>
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its binding with NO. In the absence of NO NorR behaves as a repressor. The operator is either an esabox, for AHL sensing,
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        </div>
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or LldO, for lactate sensing. These operators are repressed by EsaR and LldR respectively and repression is relieved
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    <div>
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upon binding of AHL or lactate respectively (see <a href="#fig3">Fig. 3</a>). The modular nature of our system allowed
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            <div class="image_box full_size">
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us to easily exchange the esabox with LldO, through one site-directed mutagenesis.
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                <a href="https://2016.igem.org/File:T--ETH_Zurich--ModularView.svg">
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</p>
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                    <img src="https://static.igem.org/mediawiki/2016/d/dd/T--ETH_Zurich--ModularView.svg">
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                </a>
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                <p><b>Figure 2:</b> System Overview</p>
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            </div>
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<p>
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<i>figure 2</i>
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</p>
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            <div class="sec light_grey">
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<p>
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                <h2>Inputs</h2>
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The switch module, under the control of the AND gate, is responsible for flipping a reporter cassette irreversibly. We have
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                <p>
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designed three different switches, based on integrases<sup><a href="#references">[3]</a></sup>, CRISPR/Cas9<sup><a href="#references">[4]</a></sup> and a novel genetic switch based on the recently
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                    We need a NO sensor and a AHL sensor. Later we will also need a Lactate sensor
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discovered CRISPR/Cpf1<sup><a href="#references">[5]</a></sup>
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                </p>
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</p>
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            </div>
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            <div class="sec light_grey">
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<p>
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                <h3>Switch</h3>
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The reporter module can drive expression of two different fluorescent proteins. In its default OFF state, i.e. prior to AND
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                <p> When both NO and AHL are present the hybrid promoter is activated and lead to Bxb1 invertase protein production.
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gate activation, the module expresses the orange fluorescent protein mNectarine under control of an operator (esabox
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                    This then activates our irreversible switch
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or LldO). Upon successful flipping, activation of the operator results in GFP expression (see <a href="#fig3">Fig. 3</a>).
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                </p>
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This mechanism is what makes multiplexing possible. Note that several reporter plasmids are present in the cell and the
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            </div>
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fraction of flipped cassettes can give us quantitative information on the signal intensity of the inputs activating the
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AND gate (link to switch modeling page).
 +
</p>
  
 +
<p>
 +
We chose <i>E. coli</i> as a host organism because it is easy to manipulate and engineer. In future the system could
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be transferred into a <i>Lactobacillus acidophilus</i> or <i>Lactococcus lactis</i> host, which are regarded as safe
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to ingest, this was <a href="https://2016.igem.org/Team:ETH_Zurich/Human_Practices">recommended</a> to us by Prof. Gerhard Rogler.
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</p>
  
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<div class="image_box full_size" id="fig3">
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<a href="https://static.igem.org/mediawiki/2016/3/32/T--ETH_Zurich--GeneticCircuit.png">
 +
<img src="https://static.igem.org/mediawiki/2016/3/32/T--ETH_Zurich--GeneticCircuit.png">
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</a>
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<p><b>Fig. 3:</b>Structure of our genetic circuit for IBD diagnosis. Note how NO and AHL are both necessary to express a
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molecular switch (here: Bxb1) which in turn is responsible for flipping a reporter cassette to its ON state, i.e. GFP
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can be expressed upon induction with AHL. Note that failure to activate the AND gate would result in mNectarine expression
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upon induction with AHL.</p>
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</div>
 +
</div>
 +
</div>
  
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            <div class="sec light_grey">
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<div class="sec white" id="conclusions">
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                <h3>Reporter</h3>
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<div>
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                <p> Once the switch is activated, and when AHL is present, GFP is produced.
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<h2>CONCLUSION</h2>
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                </p>
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<p>Our design for Pavlov's coli creates a novel investigation tool for IBD that:</p>
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            </div>
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<ul>
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<li>Does not disrupt the gut microbiome</li>
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<li>Can associate potential new markers of IBD pathogenesis to an established marker of IBD and memorize the event</li>
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<li>Allows for multiplexing</li>
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<li>Is flexible and easily adjustable to new potential markers due to its modular nature.</li>
 +
</ul>
 +
</div>
 +
</div>
  
 +
<div class="sec white" id="references">
 +
<div>
 +
<h2>REFERENCES</h2>
 +
            <ul>
 +
    <li>[1] G. Kolios, V. Valatas, S. G. Ward, Immunology <b>2004</b>, 113, 427-437.<br/> [2] M. Schuster, D. J. Sexton, S. P.
 +
    <li>Diggle, E. P. Greenberg, Annu Rev Microbiol <b>2013</b>, 67, 43-63.<br/> [3] J. Bonnet, P. Subsoontorn, D. Endy, Proc
 +
    <li>Natl Acad Sci U S A <b>2012</b>, 109, 8884-8889.<br/> [4] H. Huang, C. Chai, N. Li, P. Rowe, N. P. Minton, S. Yang, W.
 +
    <li>Jiang, Y. Gu, ACS Synth Biol <b>2016</b>.<br/> [5] B. Zetsche, J. S. Gootenberg, O. O. Abudayyeh, I. M. Slaymaker, K.
 +
    <li>S. Makarova, P. Essletzbichler, S. E. Volz, J. Joung, J. van der Oost, A. Regev, E. V. Koonin, F. Zhang, Cell <b>2015</b>, 163, 759-771.<br/>
 +
            </ul>
 +
</div>
 +
</div>
 +
</body>
  
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    <div class="sec white" id="geneticcircuit">
 
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        <div>
 
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            <h1>Genetic circuit</h1>
 
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        </div>
 
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        <div>
 
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            <div class="image_box full_size" style="max-width: 900px;">
 
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                <a href="https://2016.igem.org/File:T--ETH_Zurich--GeneticCircuit">
 
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                    <img src="https://static.igem.org/mediawiki/2016/e/e5/T--ETH_Zurich--GeneticCircuit.svg">
 
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                </a>
 
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                <p><b>Figure 3:</b> Genetic Circuit</p>
 
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            </div>
 
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<p>
 
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<i>figure 3</i>
 
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</p>
 
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            <div class="sec white">
 
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                <h2>NO sensor</h2>
 
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                <p>
 
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                    We use here the PnorV promoter, specific to NO sensing. We also use a constitutively produced NorR protein. NorR exist under
 
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                    a dimer form in the cell. Each dimer is able to bind to the 3 binding site present on PnorV. Then those
 
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                    three dimer assemble in a hexamric ring like structure. When NO is present in the medium, it binds to
 
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                    the structure and activate the promoter.
 
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                </p>
 
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            </div>
 
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            <div class="sec white">
 
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                <h2>AHL sensor</h2>
 
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                <p>
 
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                    In addition to the PnorV promoter, we had downstream esaboxes. Esar is constituvely produced in our bacteria. Just like NorR
 
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                    it exists as a dimer inb the cell. The dimer form binds to the esaboxes forming a roadblock and unabling
 
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                    gene transcription when NO only is present. When AHL enter the cell it binds to the EsaR dimer and free
 
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                    the promoter, allowing transcription.
 
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                </p>
 
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            </div>
 
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            <div class="sec white">
 
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                <h2>Lactate sensor</h2>
 
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                <p>
 
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                    Recent studies highlighted the fact that Lactate seems to be overpresent in some very heavy case of IBD, expecially in children.
 
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                    Thus it is also intersting yto investigate the role of Lactate in IBD occurences. We uses here a modified
 
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                    version of the Plac promoter. two LldR binding sites O1 and O2 are situated upstream the promoter. LldR
 
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                    and LldD (Lactate -> Pyruvate catalysist) are constitutively produced. in absence of Lacatte, LldR binds
 
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                    to O1 and O2 forming a DNA loop and preventing transcription. When Lactate enter the system, it binds
 
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                    to tyhe LldR dimer and free the promoter. We introduced LldD to increase the threshold of Lactate sensing.
 
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                </p>
 
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            </div>
 
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            <div class="sec white">
 
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                <h2>AND GATE</h2>
 
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                <p>
 
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                    The AND gate is the association of both NO sensor and AHL or Lactate sensor. It is constituted by a hybrid promoter composed
 
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                    of the PnorV promoter and the downstream esaboxes.
 
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                </p>
 
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            </div>
 
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            <div class="sec white">
 
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                <h2>Switch Module</h2>
 
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                <p>
 
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                    AND gate activation triggers Bxb1 production. Bxb1 is an invertase protein. Its role is to inverse the DNA strand containing
 
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                    the GFP gene.
 
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                </p>
 
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            </div>
 
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            <div class="sec white">
 
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                <h2>Reporter Module</h2>
 
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Latest revision as of 03:41, 20 October 2016

PAVLOV'S COLI

DESIGN

OVERVIEW

Inflammatory bowel disease (IBD) is an insufficiently understood chronic disease, whose increasing incidence, lack of proper treatment and diagnostic methods make it an urgent problem to solve[1]. The current methods for diagnosing IBD mainly rely on endoscopies, which are invasive and do not allow to properly characterize the microbiome of patients, believed to be implicated in pathogenesis.

We designed an alternative solution for in vivo diagnosis, using hydrogel-encapsulated Pavlov's coli. In order for Pavlov's coli to efficiently gather information on important aspects of IBD, such as inflammation and microbiome composition, we designed our system such that it consists of:

  1. A biological AND gate that allows our system to associate known IBD markers present in the gut.
  2. A switch acting as memory, which is irreversibly flipped from "OFF" to "ON" if both markers are simultaneously detected by the AND gate. This switching mechanism renders a reporter (GFP, see later) sensitive to one of the two biomarkers previously encountered by our logic AND gate.
  3. Information sensed by the AND gate is stored (remembered) by the reporter via the switching mechanism and can be read-out later in a laboratory. This conditional activation of our reporter justifies the name of our diagnostic tool - Pavlov's coli.

The encapsulation should allow for diffusion of molecules, while preventing our bacteria from colonizing the gut, therefore providing a non-invasive tool, which does not disrupt the microbiome.

Fig. 1: Structure of Pavlov's coli's genetic circuit. (1) The sensor module comprises an AND gate which associates an inflammation marker with a candidate marker, representative of microbiome composition. (2) The switch module consists of an irreversible genetic switch. (3) The reporter module can be used to retrieve information about the biomarkers sensed by our AND gate.

An additional feature of our system is that it allows for multiplexing, meaning processing multiple signals through one channel: in our system, the reporter module (3) is activated by one of the two markers also required to activate the sensor module, i.e. the AND gate (1) (see Fig. 1). The architecture of our system has the following implication: a suite of bacterial populations, each responsive to different IBD-relevant candidate markers (e.g. different AHL molecules, lactate, etc.) as well as to an inflammation marker (e.g. NO), can be engineered, complexed together and administered to a patient; upon recovery, re-exposure of faecal samples to a previously encountered biomarker can be read out through our reporter module (see Fig. 2).

Fig. 2:Mode of action of Pavlov's coli. Step 1: After administration of our bacteria, biological markers can be sensed in the gut through a logical AND gate; the event is memorized through a switching mechanism. Step 2: Following excretion and retrieval of our bacteria, the memory can be read-out by exposing the samples containing our bacteria to a host of IBD-relevant candidate markers.

Therefore, Pavlov's coli holds promise as a non-invasive, modular, multipleable, in vivo diagnostic tool for IBD investigation.

Sensing in the gut

One major challenge in synthetic biology is the difficulty to combine modular frameworks into higher order networks that remain reliable. We designed our circuit as interchangeable, easily tunable parts, each of which is backed by a computer model and experimental data.

IBD is normally accompanied by severe inflammation of the intestine. Several sources in the literature report on a sharp increase in Nitric Oxide (NO) levels in inflamed areas. Based on this, we chose to incorporate a sensor for NO as part of a logic AND gate in our engineered bacteria[1].

Along with the presence of NO, Pavlov's coli's logic gate requires a second input in order to activate an irreversible switch (see Fig. 1; also, see section below). As the second input, we chose a marker which can provide information on microbiome composition: AHL. A number of AHLs have been found to be informative as to specific functions (e.g. biofilm formation) and microbiome composition[2].

In an alternative design, we chose to replace the AHL sensor with lactate sensor. In an interview with an expert on IBD, Prof. Lacroix, we discussed the potential of bacterial metabolites as an alternative microbiome marker. One such a candidate turned out to be lactate, which is produced and metabolized by different bacteria in the gut. Although it is not as informative as AHL to the end of microbiome characterization, there is a strong interest in developing methods for lactate detection in the gut. Due to its nature as an intermediate metabolite, it is currently not possible to measure lactate concentrations in fecal samples of patients, adding appeal to the development of Pavlov's coli.

DETAILED IMPLEMENTATION

Our system is composed of a logic AND gate, a switch and a reporter, behaving together as an associative learning circuit.

The AND gate is composed of the PnorV promoter and an operator. The promoter can be activated by NorR upon its binding with NO. In the absence of NO NorR behaves as a repressor. The operator is either an esabox, for AHL sensing, or LldO, for lactate sensing. These operators are repressed by EsaR and LldR respectively and repression is relieved upon binding of AHL or lactate respectively (see Fig. 3). The modular nature of our system allowed us to easily exchange the esabox with LldO, through one site-directed mutagenesis.

The switch module, under the control of the AND gate, is responsible for flipping a reporter cassette irreversibly. We have designed three different switches, based on integrases[3], CRISPR/Cas9[4] and a novel genetic switch based on the recently discovered CRISPR/Cpf1[5]

The reporter module can drive expression of two different fluorescent proteins. In its default OFF state, i.e. prior to AND gate activation, the module expresses the orange fluorescent protein mNectarine under control of an operator (esabox or LldO). Upon successful flipping, activation of the operator results in GFP expression (see Fig. 3). This mechanism is what makes multiplexing possible. Note that several reporter plasmids are present in the cell and the fraction of flipped cassettes can give us quantitative information on the signal intensity of the inputs activating the AND gate (link to switch modeling page).

We chose E. coli as a host organism because it is easy to manipulate and engineer. In future the system could be transferred into a Lactobacillus acidophilus or Lactococcus lactis host, which are regarded as safe to ingest, this was recommended to us by Prof. Gerhard Rogler.

Fig. 3:Structure of our genetic circuit for IBD diagnosis. Note how NO and AHL are both necessary to express a molecular switch (here: Bxb1) which in turn is responsible for flipping a reporter cassette to its ON state, i.e. GFP can be expressed upon induction with AHL. Note that failure to activate the AND gate would result in mNectarine expression upon induction with AHL.

CONCLUSION

Our design for Pavlov's coli creates a novel investigation tool for IBD that:

  • Does not disrupt the gut microbiome
  • Can associate potential new markers of IBD pathogenesis to an established marker of IBD and memorize the event
  • Allows for multiplexing
  • Is flexible and easily adjustable to new potential markers due to its modular nature.

REFERENCES

  • [1] G. Kolios, V. Valatas, S. G. Ward, Immunology 2004, 113, 427-437.
    [2] M. Schuster, D. J. Sexton, S. P.
  • Diggle, E. P. Greenberg, Annu Rev Microbiol 2013, 67, 43-63.
    [3] J. Bonnet, P. Subsoontorn, D. Endy, Proc
  • Natl Acad Sci U S A 2012, 109, 8884-8889.
    [4] H. Huang, C. Chai, N. Li, P. Rowe, N. P. Minton, S. Yang, W.
  • Jiang, Y. Gu, ACS Synth Biol 2016.
    [5] B. Zetsche, J. S. Gootenberg, O. O. Abudayyeh, I. M. Slaymaker, K.
  • S. Makarova, P. Essletzbichler, S. E. Volz, J. Joung, J. van der Oost, A. Regev, E. V. Koonin, F. Zhang, Cell 2015, 163, 759-771.

Thanks to the sponsors that supported our project: