What is iron for ? Fe 2+ Ferrous iron 2 What is iron for ? Fe 2+ - - PowerPoint PPT Presentation

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What is iron for ? Fe 2+ Ferrous iron 2 What is iron for ? Fe 2+ - - PowerPoint PPT Presentation

Feb 01, 2024 380 likes •1.31k views

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

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What is iron for ?

2

Fe2+

Ferrous iron

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What is iron for ?

2

Fe2+

Ferrous iron Heme

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What is iron for ?

2

Fe2+

Oxygen carrier Ferrous iron Heme

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What is iron for ?

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Fe2+

Oxygen carrier Cellular energy production Ferrous iron Heme

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What is iron for ?

2

Fe2+

Oxygen carrier Cellular energy production Ferrous iron

Iron coli Project Iron related diseases

Heme

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Hemochromatosis

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Hemochromatosis

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2 200 000 cases in Europe

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Hemochromatosis

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2 200 000 cases in Europe Overabsorption of iron

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Hemochromatosis

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2 200 000 cases in Europe Overabsorption of iron Permanent insufficiencies and fatigue

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Hemochromatosis

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2 200 000 cases in Europe Overabsorption of iron Permanent insufficiencies and fatigue Treatment: Blood-letting

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Our strategy

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Our strategy

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Our strategy

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Sensing Analyzing Neutralizing

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Our strategy

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Our strategy

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Our strategy

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Our strategy

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Our strategy

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Our goal

Alternative treatment to blood-lettings

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Chelating the iron in the intestine

Sensor Inverter Chelator

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Enterobactins to chelate them all

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Ka = 1039 M−1 Enterobactin structure Natural production when [Fe2+] < 10-7 M

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Human Practice

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Patients response to Iron Coli treatment

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Would you take our treatment if it was allowed by the national agency for drug safety ?

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Patients response to Iron Coli treatment

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« Would you take our treatment if it was allowed by the national agency for drug safety ? »

N = 270 answers

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Potential consequences of Iron Coli

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Fault Tree

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Potential consequences of Iron Coli

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Fault Tree

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Model

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Is Flush strategy viable ?

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Can Iron coli prevent iron absorption ?

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Is Flush strategy viable ?

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Can Iron coli prevent iron absorption ?

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Is Flush strategy viable ?

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Can Iron coli prevent iron absorption ?

ODE Model Assumption: Instantaneous enterobactin production

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Exploring flush strategy

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Iron absorption Iron in duodenum

Without treatment Without treatment

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Exploring flush strategy

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Iron absorption Iron in duodenum

Without treatment With treatment Without treatment With treatment

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Exploring flush strategy

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42 sec. flush: Iron absorption is divided by 2

Iron absorption Iron in duodenum

Without treatment With treatment Without treatment With treatment

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Less frequent blood-lettings beneficial to working patients

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Would you take our treatment if it’s allowed by the national agency for drug safety ?

N = 270 answers

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Less frequent blood-lettings beneficial to working patients

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Would you take our treatment if it’s allowed by the national agency for drug safety ?

N = 270 answers

Bloodletting is very time-consuming

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Biology

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Project overview

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Sensor Inverter Chelator Iron coli

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Harnessing FUR for iron sensing

Sensor system

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Extracted promoters:

  • pAceB
  • pFepA
  • pFes
  • pYncE
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Harnessing FUR for iron sensing

Sensor system

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Extracted promoters:

GFP pAceB T RBS FUR Fe2+

Absence of iron

  • pFes
  • pYncE
  • pAceB
  • pFepA
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Harnessing FUR for iron sensing

Sensor system

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Extracted promoters:

GFP pAceB T RBS FUR Fe2+

Absence of iron

GFP pAceB T RBS FUR Fe2+

Presence of iron

  • pFes
  • pYncE
  • pAceB
  • pFepA
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Characterisation of iron sensing device

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Cultures in varying iron concentration No significant reduction of growth rate

(h)

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Characterisation of iron sensing device

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Cultures in varying iron concentration No significant reduction of growth rate repressed by iron

(h)

BBa_K1163102 W

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Inverting the signal

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 Activation

FUR

𝐆𝐟𝟑+ LacI T RBS T RBS RFP

Presence of iron

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Inverting the signal

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 Activation

FUR

𝐆𝐟𝟑+ LacI T RBS T RBS RFP

Presence of iron

FUR

LacI T RBS

No Activation

LacI

T RBS RFP

Absence of iron

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Characterisation of iron sensing device

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Cultures in varying iron concentration No significant reduction of growth rate

(h)

RFP/D.O.

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Characterisation of iron sensing device

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Cultures in varying iron concentration No significant reduction of growth rate inverter and reporter – the system expresses RFP as a function of iron concentration K1163103

(h)

J04450

RFP/D.O.

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Enterobactin biosynthesis pathway

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6 enzymes: EntA, EntB, EntC, EntD, EntE, EntF

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Genome scale metabolic model

Can our construct unbalance E. coli metabolism?

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Genome scale metabolic model

Can our construct unbalance E. coli metabolism?

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Genome scale metabolic model

Can our construct unbalance E. coli metabolism?

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Genome scale metabolic model

Can our construct unbalance E. coli metabolism?

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  • E. Coli iJR904 + 1 reaction (ENTSYNTH)

Flux Balance Analysis framework

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FBA simulations

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Need to add chorismate to the medium

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The chelating device

Design 1: Fully refactored enterobactin operon

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Design 2: Semi-refactored operon

EntB EntC EntE pLac EntA EntD EntF pLac

Amp Kan

EntC EntE EntB pLac EntA EntD EntF EntH

Tet

All individual parts are ready

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Enterobactin production kinetics

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How much time from Iron Sensing to enterobactin production?

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Enterobactin production kinetics

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How much time from Iron Sensing to enterobactin production?

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Enterobactin production kinetics

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How much time from Iron Sensing to enterobactin production?

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Enterobactin production kinetics

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How much time from Iron Sensing to enterobactin production?

ODE Model Inverter system + Chemical kinetics Parameters → literature

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Interpretation of simulations

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Interpretation of simulations

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Iron Enzymes Enterobactin Time (min)

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Interpretation of simulations

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Enterobactin production after 5 hours

Iron Enzymes Enterobactin Time (min)

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Interpretation of simulations

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Enterobactin production after 5 hours

Need to rethink flush approach

Iron Enzymes Enterobactin Time (min)

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Capsule

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Iron coli needs time to act

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How to give Iron coli enough time to produce enterobactins ?

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Iron coli needs time to act

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How to give Iron coli enough time to produce enterobactins ?

Stomach Upper intestine

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Improving galenic formulation for bacteria

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Improving galenic formulation for bacteria

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Slowed down flush strategy

Viscous matrix retains Iron coli in the upper intestine

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HPMC matrix Intestinal barrier

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Capsule manufacturing

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Capsule manufacturing

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Capsule manufacturing

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Capsule follows the actual norms

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Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule

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Capsule follows the actual norms

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Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule

pH = 2 (Stomach)

No dissolution after 2 hours

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Capsule follows the actual norms

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Requirements of European Pharmacopeoa for a gastro-enteric resistant capsule

pH = 7 (Upper intestine)

Dissolution before 1 hour

pH = 2 (Stomach)

No dissolution after 2 hours

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Gathering patients, experts and the team

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We organised a seminar with researchers, clinicians, associations of patients, patients, and our iGEM Evry Team

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Confronting our project to reality

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What we want/need to do How we achieve it Result

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Confronting our project to reality

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What we want/need to do How we achieve it Result Ideal situation

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Confronting our project to reality

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Human goal

Helping hemochromatosis patients

What we want/need to do How we achieve it Result Ideal situation

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Confronting our project to reality

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Human goal

Helping hemochromatosis patients

Means

Synthetic biology

What we want/need to do How we achieve it Result Ideal situation

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Confronting our project to reality

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Human goal

Helping hemochromatosis patients

Means

Synthetic biology

Project

Iron coli

What we want/need to do How we achieve it Result Ideal situation

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Confronting our project to reality

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Concrete iGEM situation What we want/need to do How we achieve it Result

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Confronting our project to reality

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Concrete iGEM situation What we want/need to do How we achieve it Result

Means

Synthetic biology

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Confronting our project to reality

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Concrete iGEM situation What we want/need to do How we achieve it Result

Means

Synthetic biology

Project

Iron coli

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Confronting our project to reality

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Concrete iGEM situation What we want/need to do How we achieve it Result

Means

Synthetic biology

Project

Iron coli

Human goal

Helping hemochromatosis patients

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Philosophical issue

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We want to apply synthetic biology to reduce the suffering of hematochromatosis patients Are we using patients to prove the usefulness of synthetic biology ?

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Achievements

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We built and characterised 8 biobricks

  • Several iron sensing systems
  • A working signal inverter

K1163102 K1163103

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Achievements

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We built and characterised 8 biobricks

  • Several iron sensing systems
  • A working signal inverter

K1163102 K1163103

We created an original delivery capsule

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Achievements

36

We built and characterised 8 biobricks

  • Several iron sensing systems
  • A working signal inverter

K1163102 K1163103

We created an original delivery capsule Modelling helped many steps of the design

  • ODE model for disease and the treatment in the gut
  • Kinetic equations model of enterobactin synthesis
  • FBA model for determining metabolism interactions
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Achievements

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We built and characterised 8 biobricks

  • Several iron sensing systems
  • A working signal inverter

K1163102 K1163103

We made an integrated human practice study

  • Patient survey and opinion
  • Seminar Doctor/Patient/Researcher
  • Constructed a fault tree
  • Philosophical study on goals and means in SynBio

We created an original delivery capsule Modelling helped many steps of the design

  • ODE model for disease and the treatment in the gut
  • Kinetic equations model of enterobactin synthesis
  • FBA model for determining metabolism interactions
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Coming soon

Finalise cloning of enterobactin genes

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Coming soon

Future characterisation (Brian C. Louden et al., 2011)

Finalise cloning of enterobactin genes Characterise their production

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Sponsors

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