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| − | <div class="sec white" id="abstractview"> | + | <div class="sec white" id="safelabwork"> |
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| − | <div class="sec light_grey" id="systemoverview"> | + | <div class="sec light_grey" id="safetyofcurrentprojectdesign"> |
| | <div> | | <div> |
| − | <h1>SYSTEM OVERVIEW</h1> | + | <h1>SAFETY IN OUR CURRENT PROJECT DESIGN</h1> |
| − | </div>
| + | |
| | <div> | | <div> |
| − | <div class="image_box full_size">
| + | </div> |
| − | <a href="https://2016.igem.org/File:T--ETH_Zurich--ModularView.svg">
| + | The goal of our project in the scope of iGEM until the giant jamboree is to design and show that our system works under controlled experimental conditions in the laboratory which would mimic real life conditions. Thus we have no intention to release bacteria with our current design in the environment or to consume it. In our project we chose to work with several different well known lab strains of E. coli: TOP10, DH5alpha, Keio strains and EcNR1. They are all derivatives of K12 E. coli strain and belong to the biosafety level 1. Biosafety level 1 organisms pose little risk to the researcher and the environment. However, we are working with GMO strains which cannot be released into the environment and low risk does not equal zero risk. For this reason we stick to all safety regulations for biosafety level 1 laboratory, such as wearing and frequently changing gloves, wearing lab coat and disinfecting the working area after the experiment. All waste that has been in contact with the bacteria is autoclaved. To mimic conditions in the gut, we chose to work with DETA/NO (as a source of nitric oxide (NO)), homoserine lactone (AHL) and lactate. We took extra precaution in handling experiments where we used DETA/NO or ganciclovir. |
| − | <img src="https://static.igem.org/mediawiki/2016/d/dd/T--ETH_Zurich--ModularView.svg">
| + | </div> |
| − | </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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| − | | + | |
| − | <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>
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| − | 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.
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| − | </p>
| + | |
| − | </div>
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| − | | + | |
| − | <div class="sec light_grey">
| + | |
| − | <h3>SWITCH</h3>
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| − | <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>.
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| − |
| + | |
| − | </p>
| + | |
| − | </div>
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| − | | + | |
| − | | + | |
| − | | + | |
| − | <div class="sec light_grey">
| + | |
| − | <h3>REPORTER</h3>
| + | |
| − | <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.
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| − | </p>
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| − | </div>
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| − | | + | |
| − | | + | |
| − | </div>
| + | |
| | </div> | | </div> |
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| − | <div class="sec white" id="geneticcircuit"> | + | <div class="sec white" id="safetyoffinalprojectdesign"> |
| | <div> | | <div> |
| − | <h1>GENETIC CIRCUIT</h1> | + | <h1>SAFETY FEATURES OF OUR FINAL PROJECT DESIGN</h1> |
| | </div> | | </div> |
| − | <div> | + | <div> |
| − | | + | |
| − | <div class="image_box full_size" style="max-width: 900px;">
| + | |
| − | <a href="https://2016.igem.org/File:T--ETH_Zurich--GeneticCircuit">
| + | |
| − | <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>
| + | |
| − | <p>
| + | |
| − | <i>figure 3</i>
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| − | </p>
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| | <div class="sec white"> | | <div class="sec white"> |
| − | <h2>NO SENSOR</h2>
| |
| | <p> | | <p> |
| − | 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 | + | The final goal of our project is to design a bacteria-based detection system to simultaneously detect compounds associated with inflammation and microbiota in the gut of an inflammatory bowel disease (IBD) patient. This requires ingestion of our device by the patient. Our bacteria will travel through the human digestive system in a capsule. The encapsulated bacteria will be collected from the feces and will be analyzed in the lab. The administration and recovery of our bacterial device would be done by trained medical personnel. There are several safety risks which we need to consider in the design of our final device. |
| − | 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 <i>NO</i> is present in the medium, it binds to
| + | |
| − | the structure and activate the promoter.
| + | |
| | </p> | | </p> |
| | </div> | | </div> |
| − | | + | </div> |
| − | <div class="sec white">
| + | |
| − | <h2>AHL SENSOR</h2> | + | |
| − | <p>
| + | |
| − | 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 in the cell. The dimer form binds to the esaboxes forming a roadblock and unabling
| + | |
| − | 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.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="sec white">
| + | |
| − | <h2>LACTATE SENSOR</h2>
| + | |
| − | <p>
| + | |
| − | 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 <i>LldR</i> binding sites O1 and O2 are situated upstream the promoter. <i>LldR</i>
| + | |
| − | 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 the <i>LldR</i> dimer and free the promoter. We introduced <i>LldD</i> to increase the threshold of Lactate sensing.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="sec white">
| + | |
| − | <h2>AND GATE</h2>
| + | |
| − | <p>
| + | |
| − | 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. Model shows that playing with <i>NorR</i> and <i>EsaR</i> production and degradation rates can allow a fine tuning of the sensor to adapt to the wished detection range.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="sec white">
| + | |
| − | <h2>SWITCH MODULE</h2>
| + | |
| − | <p>
| + | |
| − | AND gate activation triggers integrase production. Its role is to inverse the DNA strand containing
| + | |
| − | the GFP gene.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | <div class="sec white">
| + | |
| − | <h2>REPORTER MODULE</h2>
| + | |
| − | <p>
| + | |
| − | the reporter module is just constituted of some esaboxes and the GFP gene. Under AHL presence, the reporter (GFP) is expressed.
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | <div class="sec light_grey" id="conclusion">
| + | |
| − | <div>
| + | |
| − | <h1>CONCLUSION</h1>
| + | |
| − | <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 as interchangeable, easily tuneable parts. This allows multiplexing, one of our circuit’s major advantages for a non-invasive, modular, multiplexing, in vivo tool to investigate IBD’s unknown causes.
| + | |
| − | | + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | </div>
| + | |
| − | | + | |
| − |
| + | |
| | | | |
| | </body> | | </body> |
SAFE LAB WORK
Safety is an essential part of any lab project in science. Proper safety regulations and precautions lower the risk of accidents. They are designed to protect the experimenter, the project, the public and the environment. At ETH-DBSSE it is mandatory for every person to take a Safety Course. The course is held by Mr. Niels Buerckert, who is the Technical Manager and head of the department’s Safety Committee. During the Safety Course we first covered basic safety measurements required for both office and lab members of the department. We were instructed how to handle hazardous situations in case of natural disaster or accident. We got acquainted with locations of fire extinguishers, fire blankets and emergency exits. In the case of a small fire, the fire blanket is used. In the case of a bigger fire, the CO2 fire extinguisher is used. If paper or carton is on fire, foam extinguisher is used instead. We were instructed who to call in the case of fire and how to act and who to call in the case of an injury. After we considered different specific situations in basic safety training, we covered lab safety measurements and regulations.
Lab safety measurements tackle every aspect of lab work. The experimenter has to wear proper clothing which covers as much skin as possible. Shorts and open shoes are not allowed. Additionally the experimenter needs to wear a lab coat, gloves and wear goggles on the top of the head. Food and drinks are not allowed in the lab. Smoking is forbidden. We were instructed how to properly label hazardous chemicals and how to act in the case we encounter unfamiliar or unlabeled chemical. In the case where we encounter unknown chemical in solid form we use a brush to gently remove the chemical and dispose it in hazardous waste. Any unlabeled liquid is considered as a potential hazardous chemical. We got acquainted with the specifics of waste management in this building: the location of clean room, how to properly transport chemicals from one part of the building to another, how to prevent potential spread of chemicals from our lab to other areas of the building. Chemicals need to be transported in the chemical bucket. When transferring chemicals from one floor to another, they are not allowed in the personel elevator. The experimenter must send the chemical separately in the elevator for chemicals, while the experimenter uses the personel elevator. Gloves need to be removed after exiting the lab. If the chemicals require handling in gloves, the experimenter must use one hand without a glove to interact with the surroundings (opening doors etc) and one hand with a glove to carry the chemicals. We were taught how to properly maintain a clean lab space and how to correctly store chemicals in the lab to prevent accidents. Every sink has an eye shower and every floor has emergency showers in case a person gets in contact with a hazardous chemical.
SAFETY IN OUR CURRENT PROJECT DESIGN
The goal of our project in the scope of iGEM until the giant jamboree is to design and show that our system works under controlled experimental conditions in the laboratory which would mimic real life conditions. Thus we have no intention to release bacteria with our current design in the environment or to consume it. In our project we chose to work with several different well known lab strains of E. coli: TOP10, DH5alpha, Keio strains and EcNR1. They are all derivatives of K12 E. coli strain and belong to the biosafety level 1. Biosafety level 1 organisms pose little risk to the researcher and the environment. However, we are working with GMO strains which cannot be released into the environment and low risk does not equal zero risk. For this reason we stick to all safety regulations for biosafety level 1 laboratory, such as wearing and frequently changing gloves, wearing lab coat and disinfecting the working area after the experiment. All waste that has been in contact with the bacteria is autoclaved. To mimic conditions in the gut, we chose to work with DETA/NO (as a source of nitric oxide (NO)), homoserine lactone (AHL) and lactate. We took extra precaution in handling experiments where we used DETA/NO or ganciclovir.
SAFETY FEATURES OF OUR FINAL PROJECT DESIGN
The final goal of our project is to design a bacteria-based detection system to simultaneously detect compounds associated with inflammation and microbiota in the gut of an inflammatory bowel disease (IBD) patient. This requires ingestion of our device by the patient. Our bacteria will travel through the human digestive system in a capsule. The encapsulated bacteria will be collected from the feces and will be analyzed in the lab. The administration and recovery of our bacterial device would be done by trained medical personnel. There are several safety risks which we need to consider in the design of our final device.
Thanks to the sponsors that supported our project:
