2 What is iron for ? Fe 2+ Ferrous iron
2 What is iron for ? Fe 2+ Ferrous iron Heme
2 What is iron for ? Fe 2+ Ferrous iron Heme Oxygen carrier
2 What is iron for ? Fe 2+ Ferrous iron Heme Oxygen carrier Cellular energy production
2 What is iron for ? Fe 2+ Ferrous iron Heme Oxygen carrier Cellular energy production Iron coli Project Iron related diseases
3 Hemochromatosis
3 Hemochromatosis 2 200 000 cases in Europe
3 Hemochromatosis 2 200 000 cases in Europe Overabsorption of iron
3 Hemochromatosis 2 200 000 cases in Europe Overabsorption of iron Permanent insufficiencies and fatigue
3 Hemochromatosis 2 200 000 cases in Europe Overabsorption of iron Permanent insufficiencies and fatigue Treatment: Blood-letting
4 Our strategy
4 Our strategy
4 Our strategy Analyzing Sensing Neutralizing
4 Our strategy
4 Our strategy
4 Our strategy
4 Our strategy
4 Our strategy
6 Our goal Alternative treatment to blood-lettings Sensor Inverter Chelator Chelating the iron in the intestine
7 Enterobactins to chelate them all Enterobactin structure K a = 10 39 M −1 Natural production when [Fe 2+ ] < 10 -7 M
8 Human Practice
9 Patients response to Iron Coli treatment Would you take our treatment if it was allowed by the national agency for drug safety ?
9 Patients response to Iron Coli treatment « Would you take our treatment if it was allowed by the national agency for drug safety ? » N = 270 answers
10 Potential consequences of Iron Coli Fault Tree
10 Potential consequences of Iron Coli Fault Tree
11 Model
12 Is Flush strategy viable ? Can Iron coli prevent iron absorption ?
12 Is Flush strategy viable ? Can Iron coli prevent iron absorption ?
12 Is Flush strategy viable ? Can Iron coli prevent iron absorption ? ODE Model Assumption: Instantaneous enterobactin production
13 Exploring flush strategy Iron absorption Without treatment Iron in duodenum Without treatment
13 Exploring flush strategy Iron absorption Without treatment With treatment Iron in duodenum Without treatment With treatment
13 Exploring flush strategy Iron absorption Without treatment With treatment Iron in duodenum Without treatment With treatment 42 sec. flush: Iron absorption is divided by 2
14 Less frequent blood-lettings beneficial to working patients Would you take our treatment if it’s allowed by the national agency for drug safety ? N = 270 answers
14 Less frequent blood-lettings beneficial to working patients Would you take our treatment if it’s allowed by the national agency for drug safety ? N = 270 answers Bloodletting is very time-consuming
15 Biology
16 Project overview Iron coli Inverter Sensor Chelator
17 Harnessing FUR for iron sensing Sensor system o pAceB o pFes Extracted promoters : o pFepA o pYncE
17 Harnessing FUR for iron sensing Sensor system o pAceB o pFes Extracted promoters : o pFepA o pYncE Fe 2+ FUR pAceB RBS GFP T Absence of iron
17 Harnessing FUR for iron sensing Sensor system o pAceB o pFes Extracted promoters : o pFepA o pYncE FUR Fe 2+ FUR Fe 2+ pAceB pAceB RBS GFP T RBS GFP T Absence of iron Presence of iron
18 Characterisation of iron sensing device Cultures in varying iron concentration No significant reduction of growth rate (h)
18 Characterisation of iron sensing device Cultures in varying iron concentration No significant reduction of growth rate repressed by iron W BBa_K1163102 (h)
19 Inverting the signal Presence of iron FUR 𝐆𝐟 𝟑+ LacI T RBS RFP T RBS Activation
19 Inverting the signal Presence of iron Absence of iron FUR FUR 𝐆𝐟 𝟑+ LacI T RBS LacI T RBS LacI RFP T RBS RFP T RBS Activation No Activation
20 Characterisation of iron sensing device Cultures in varying iron concentration No significant reduction of growth rate RFP/D.O. (h)
20 Characterisation of iron sensing device Cultures in varying iron concentration No significant reduction of growth rate inverter and reporter – the system K1163103 J04450 expresses RFP as a function of iron concentration RFP/D.O. (h)
21 Enterobactin biosynthesis pathway 6 enzymes: EntA, EntB, EntC, EntD, EntE, EntF
22 Genome scale metabolic model Can our construct unbalance E. coli metabolism?
22 Genome scale metabolic model Can our construct unbalance E. coli metabolism?
22 Genome scale metabolic model Can our construct unbalance E. coli metabolism?
22 Genome scale metabolic model Can our construct unbalance E. coli metabolism? Flux Balance Analysis framework E. Coli iJR904 + 1 reaction (ENTSYNTH)
23 FBA simulations Need to add chorismate to the medium
24 The chelating device All individual parts are ready Design 1: Fully refactored enterobactin operon pLac Amp EntB EntC EntE pLac Kan EntA EntD EntF Design 2: Semi-refactored operon pLac Tet EntF EntD EntC EntE EntB EntA EntH
25 Enterobactin production kinetics How much time from Iron Sensing to enterobactin production?
25 Enterobactin production kinetics How much time from Iron Sensing to enterobactin production?
25 Enterobactin production kinetics How much time from Iron Sensing to enterobactin production?
25 Enterobactin production kinetics How much time from Iron Sensing to enterobactin production? ODE Model Inverter system + Chemical kinetics Parameters → literature
26 Interpretation of simulations
26 Interpretation of simulations Enzymes Iron Enterobactin Time (min)
26 Interpretation of simulations Enzymes Iron Enterobactin Time (min) Enterobactin production after 5 hours
26 Interpretation of simulations Enzymes Iron Enterobactin Time (min) Enterobactin production after 5 hours Need to rethink flush approach
27 Capsule
28 Iron coli needs time to act How to give Iron coli enough time to produce enterobactins ?
28 Iron coli needs time to act How to give Iron coli enough time to produce enterobactins ? Stomach Upper intestine
29 Improving galenic formulation for bacteria
29 Improving galenic formulation for bacteria
30 Slowed down flush strategy Intestinal barrier HPMC matrix Viscous matrix retains Iron coli in the upper intestine
31 Capsule manufacturing
31 Capsule manufacturing
31 Capsule manufacturing
32 Capsule follows the actual norms Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule
32 Capsule follows the actual norms Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule No dissolution after 2 hours pH = 2 (Stomach)
32 Capsule follows the actual norms Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule No dissolution after 2 hours pH = 2 (Stomach) Dissolution before 1 hour pH = 7 (Upper intestine)
33 Gathering patients, experts and the team We organised a seminar with researchers, clinicians, associations of patients, patients, and our iGEM Evry Team
34 Confronting our project to reality What we want/need to do How we achieve it Result
34 Confronting our project to reality Ideal situation What we want/need to do How we achieve it Result
34 Confronting our project to reality Ideal situation Human goal What we want/need to do Helping hemochromatosis patients How we achieve it Result
34 Confronting our project to reality Ideal situation Human goal What we want/need to do Helping hemochromatosis patients Means How we achieve it Synthetic biology Result
34 Confronting our project to reality Ideal situation Human goal What we want/need to do Helping hemochromatosis patients Means How we achieve it Synthetic biology Project Result Iron coli
34 Confronting our project to reality Concrete iGEM situation What we want/need to do How we achieve it Result
34 Confronting our project to reality Concrete iGEM situation Means What we want/need to do Synthetic biology How we achieve it Result
34 Confronting our project to reality Concrete iGEM situation Means What we want/need to do Synthetic biology Project How we achieve it Iron coli Result
34 Confronting our project to reality Concrete iGEM situation Means What we want/need to do Synthetic biology Project How we achieve it Iron coli Human goal Result Helping hemochromatosis patients
35 Philosophical issue We want to apply synthetic biology to reduce the suffering of hematochromatosis patients Are we using patients to prove the usefulness of synthetic biology ?
36 Achievements We built and characterised 8 biobricks o Several iron sensing systems K1163102 o A working signal inverter K1163103
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