Team:Evry/Project

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<h1>Overal project</h1>
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<h1>Overview</h1>
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<h2 style="text-align:center">Abstract</h2>
 
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This year, our project focuses on hematological disorders. More specifically, we focus on diseases that are subsequent to an iron overload such as hemochromatosis and thalassemia. For hereditary hemochromatosis, the symptoms are due to the overabsorption of iron from the duodenum by the mutation of the HFE protein. However, in case of thalassemia, patients excessively absorb iron because of a subsequent metabolic perturbation.
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Our Iron Coli Project deals with <a href="https://2013.igem.org/Team:Evry/Project_diseases">hemochromatosis</a>. Hemochromatosis is an autosomal recessive disorder which is the most frequent genetic disease in Europe as it affects over two millions people. It is caused by a mutation on the HFE gene, which leads to an <a href="https://2013.igem.org/Team:Evry/Project_metabolism">overabsorption of iron</a> in the upper region of the intestine. This iron overload is stored in many organs and tissues, which eventually causes chronic insuficiences as heart, liver and kidney failures.
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Nowadays, the iron overload is mainly treated by bloodlettings for hemochromatosic patients but cannot be extended to thalassemic patients who suffer from anaemia. The aim of our project is to prevent the intestinal absorption of iron, thus acting directly at the source.
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The only available treatment for patients suffering from hemochromatosic nowadays is blood-lettings. <a href="https://2013.igem.org/Team:Evry/Seminar">Discussing with patients</a> we discover that this treatment is time-consumming and is often barely tolerated. To find a solution, we worked on the creation of a bacterial treatment to prevent iron overload in hemochromatosis.</p>
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Using the Ferric Uptake Regulation (FUR) system that controls siderophore biosynthesis (iron chelator), we engineer <i>Escherichia coli</i> in order to produce these siderophores in response of high concentrations of iron. To reduce the patient's iron absorption, our bacteria will be placed in a capsule and will be ingested during a meal. Once it will arrive in the duodenum, our bacteria will produce the siderophore at their full potential and chelate the iron.
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We focused on a natural iron <a href="https://2013.igem.org/Team:Evry/Project_FUR">chelating molecule</a> called enterobactins that is produced in iron starvation case by <i>E. coli</i>. For our project, we wanted to invert this natural behaviour, so <i>E. coli</i> would produce these enterobactins in high iron concentration. To deliver our bacteria right in the duodenum where it would prevent the iron absorption, we also worked on the design of a gastro resistant <a href="https://2013.igem.org/Team:Evry/Pill_design">capsule</a>.
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<h2 style="text-align:center">Iron metabolism</h2>
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We first wanted to know if our bacterial treatment would be accepted by the patients. Then, we conducted a <a href="https://2013.igem.org/Team:Evry/HumanPractice/Patients_perceptions">survey</a> to ask for their opinion. It appears that a majority of hemochromatosic patients would actually agree on taking our treatment. <br/>
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<h2 style="text-align:center">Iron intestinal absorption by the duodenum</h2>
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We also created a <a href="https://2013.igem.org/Team:Evry/Safety">fault tree</a> to identify the consequences involved if <i>E. coli</i> will stay permantly or temporarily into the intestine. This study oriented our modelling team to a <a href="https://2013.igem.org/Team:Evry/flush_model">flush strategy</a>, meaning that <i>E. coli</i> only stays temporarily in the intestine. Their model predicted that our engineered <i>E. coli</i> (<b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b>) could divide by two the amount of iron absorbed by a hemochromatosic patient.<br/>
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Knowing that our treatment would be accepted and beneficial to patients, we then focused on the biology of our project. We obtained two biobricks : an <a href="https://2013.igem.org/Team:Evry/Sensor">iron sensing device</a> and an <a href="https://2013.igem.org/Team:Evry/Inverter">inverter signal system</a>.
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    <span align="center"> Figure 1: Intestinal iron absorption area.</span>
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    <center> Figure 2: Iron absorption by intestinal cell.</center>
 
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<h2 style="text-align:center">Related diseases</h2>
 
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<b>Hemochromatosis</b> is a genetic disease responsible for an abnormal iron overload in blood and has a occurence of 1/30000 in Europe. The illness is due to a mutation on the HFE protein which leads to a massive iron absorption by the intestins. In fact, the duodenum is insensitive to the hepcidin hormon, normally responsible for the shutdown of iron absorption. As a consequence, there is a massive entry of iron. Our body is only able to regulate the flux by shutting down its absorption. As a consequence, it starts to stock the iron in all the tissues, leading to chronic insufficiencies. Nowadays, the only effective way to treat hemochromatosis is by bleed draining. This treatment is invasive and archaic compared to the actual advances in the medical field.<br></p>
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We wonder how much time it would take for our <b><span style="color:#bb8900">Iron</span><span style="color:#7B0000"> Coli</span></b> to produce enterobactin and if overproducing them would unbalance <i>E. coli</i> metabolism. Results obtained from the <a href="https://2013.igem.org/Team:Evry/Metabolism_model">Flux Balance Analysis</a> and a <a href="https://2013.igem.org/Team:Evry/Modelmeta3">differencial equation model</a> leads our biologists to optimize their construction and design a <a href="https://2013.igem.org/Team:Evry/Pill_design">delivery capsule</a>.
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<b>Thalassemia</b> is also a genetic disease and frequently encountered around the mediterranean sea. The mutations on the hemoglobin monomers reduces the amount of oxygen transported at the scale of the red cells, thus leading to mild or severe anemia. The lack of effective red cells in the oxygen transport results in the fake signal of low iron centration in blood, thus inducting overabsorption from the duodenum. Also, since the patient all have anemia, blood withdrawal is obviously not the way to treat them. The only solutions are the use of iron chelators, with severe side effects, and blood transfusions, which ironically lead to an iron overload.
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Through our project, we managed to made an integrated <a href="https://2013.igem.org/Team:Evry/HumanPractice">human practice</a> study as well as creating useful <a href="https://2013.igem.org/Team:Evry/Modeling">models</a> that both equally contributed to the realisation of our final project.
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<h2 style="text-align:center">Our project</h2>
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Bloodletting consists of the withdrawal of blood for therapeutic purposes. It is one of the oldest medical techniques and is nowadays abandonned. However in hematology, bloodletting (also known as <i>phlebotomy</i>) seems to remain the only reliable solution to cure hemochromatosis and thalassemia. Both are genetic diseases diseases and consist of an iron overload. The iron ion is toxic due to its oxidative potential, and thus, has a very smooth regulation mechanism. In fact, the organism only absorbs the required amount of iron from the intestins. The pathogenic mechanism is due to a seemingly low iron rate in the organism and permanantly activate the absorption system by the instestins, thus leading to an iron overload. The consequences are mainly chronic insufficiencies (renal, hepatic, cardiac, ...). Despite the true efficiency of bloodletting, some patients cannot bear this archaic way of treating their disease. As a consequence, we decided to design an <i>Escherichia coli</i> capable of chelating iron in the intestins: <b>Iron Coli</b>. The bacterium will lower the amount of iron absorbed by the duodenum and reduce the frequency of bloodlettings. Also, certain patients are treated with iron chelators which have a lot of side effects. The reduction of iron absorption by Iron Coli will allow lower drug concentrations and improve the patient's comfort.<br></p>
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<h2 style="text-align:center">Targets</h2>
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<h2 style="text-align:center">Constructions</h2>
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Our goal is to lower the iron absorption from the intestins to the blood by using an iron chelating bacteria, <b>Iron Coli</b>. The objective of the labwork is to design an iron-sensible promoter with FUR and overexpress the enterobactin synthesis pathway in the presence of iron.
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Before going in the detail of our construction, we needed a iron marker in E. coli. Thus, we selected the FUR protein which negatively regulates the downstream genes. It is used in the iron regulation of the bacterium and smoothly shuts down the production ot siderophores, natural chelators of iron in the environment in iron depletion situation. When the concentration of iron exceeds 10^-6, the FUR proteins binds the ion, dimerizes and its inhibitory mechanism is activated. Below this threshold, the FUR protein's inhibition is suppressed and indirectly activates the downstream genes.
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    <center> Figure 3: Construction.</center>
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<h1 style="text-align:center">Project progression</h1>
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<h2> Part 1 </h2>
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<p>Bacteria engineering and reversed Fur system for iron sensing</p>
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<h2> Part 2 </h2>
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<p>Modeling on bacteria interaction with duodenum.</p>
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<h3> The Experiments </h3>
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<h2> Part 3 </h2>
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<h3>Results</h3>
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Latest revision as of 03:43, 29 October 2013

Iron coli project

Overview

Our Iron Coli Project deals with hemochromatosis. Hemochromatosis is an autosomal recessive disorder which is the most frequent genetic disease in Europe as it affects over two millions people. It is caused by a mutation on the HFE gene, which leads to an overabsorption of iron in the upper region of the intestine. This iron overload is stored in many organs and tissues, which eventually causes chronic insuficiences as heart, liver and kidney failures.

The only available treatment for patients suffering from hemochromatosic nowadays is blood-lettings. Discussing with patients we discover that this treatment is time-consumming and is often barely tolerated. To find a solution, we worked on the creation of a bacterial treatment to prevent iron overload in hemochromatosis.

We focused on a natural iron chelating molecule called enterobactins that is produced in iron starvation case by E. coli. For our project, we wanted to invert this natural behaviour, so E. coli would produce these enterobactins in high iron concentration. To deliver our bacteria right in the duodenum where it would prevent the iron absorption, we also worked on the design of a gastro resistant capsule.

We first wanted to know if our bacterial treatment would be accepted by the patients. Then, we conducted a survey to ask for their opinion. It appears that a majority of hemochromatosic patients would actually agree on taking our treatment.
We also created a fault tree to identify the consequences involved if E. coli will stay permantly or temporarily into the intestine. This study oriented our modelling team to a flush strategy, meaning that E. coli only stays temporarily in the intestine. Their model predicted that our engineered E. coli (Iron Coli) could divide by two the amount of iron absorbed by a hemochromatosic patient.
Knowing that our treatment would be accepted and beneficial to patients, we then focused on the biology of our project. We obtained two biobricks : an iron sensing device and an inverter signal system.

We wonder how much time it would take for our Iron Coli to produce enterobactin and if overproducing them would unbalance E. coli metabolism. Results obtained from the Flux Balance Analysis and a differencial equation model leads our biologists to optimize their construction and design a delivery capsule.

Through our project, we managed to made an integrated human practice study as well as creating useful models that both equally contributed to the realisation of our final project.