INSA-LYON Team – iGEM2014
Source: W. Eugene Smith, JAPAN. Minamata. JAPAN Mercury poisoning
Source: www.demotix.com/news/ Source: W. Eugene Smith, JAPAN. Minamata. JAPAN BANGLADESH Mercury poisoning Polluted river Buriganga in Dhaka
Source: www.demotix.com/news/ Source: rt.com/news/ Source: W. Eugene Smith, JAPAN. Minamata. JAPAN BANGLADESH CHINA Mercury poisoning Polluted river Buriganga in Polluted Water in Fuyuan Dhaka
OUR CURLY’ON PROJECT
OUR CONCEPT
OUR CONCEPT OUR FISH: Nickel
OUR CONCEPT OUR FISH: Nickel OUR HOOK: PolyhistidineTag n
OUR CONCEPT OUR FISH: Nickel OUR HOOK: PolyhistidineTag n
OUR CONCEPT OUR FISHING ROD: The curli fibers
OUR CONCEPT OUR FISHING ROD: The curli fibers Curli
BIO ENGINEERING Limited curli production Wild Type CsgA
BIO ENGINEERING Strong curli production Limited curli production Wild Type CsgA CsgA + OmpR234 CsgA
BIO ENGINEERING Strong production of Strong curli production Limited curli production modified curli CsgA modified + OmpR234 Wild Type CsgA CsgA + OmpR234 His CsgA CsgA
BIO ENGINEERING CsgA - strain BBa_K1404006
BIO ENGINEERING CsgA - strain BBa_K1404006 EX SP CsgA p70
BIO ENGINEERING CsgA - strain BBa_K1404006 EX SP CsgA CsgA p70
BIO ENGINEERING CsgA - strain BBa_K1404006 EX SP CsgA CsgA p70
BIO ENGINEERING CsgA - strain BBa_K1404006 EX SP CsgA CsgA p70
BIO ENGINEERING OmpR234 strain BBa_K1404006
BIO ENGINEERING OmpR234 strain BBa_K1404006 EX SP CsgA p70
BIO ENGINEERING OmpR234 strain BBa_K1404006 EX SP CsgA p70
BIO ENGINEERING OmpR234 strain BBa_K1404007
BIO ENGINEERING OmpR234 strain BBa_K1404007 EX SP CsgA His 1 p70
BIO ENGINEERING OmpR234 strain BBa_K1404007 EX SP CsgA His 1 CsgA His 1 p70
BIO ENGINEERING OmpR234 strain BBa_K1404007 EX SP CsgA His 1 CsgA His 1 p70
BIO ENGINEERING OmpR234 strain BBa_K1404007 EX SP CsgA His 1 CsgA His 1 p70
CsgA: THE MAIN CURLI SUBUNIT Without any Tag WILD TYPE CsgA
CsgA: THE MAIN CURLI SUBUNIT Without any Tag With one His Tag His 1 WILD TYPE CsgA CsgA His1
CsgA: THE MAIN CURLI SUBUNIT Nickel Without any Tag WILD TYPE CsgA
CsgA: THE MAIN CURLI SUBUNIT Nickel Without any Tag With one His Tag + 25 % WILD TYPE CsgA CsgA His1
CsgA: THE MAIN CURLI SUBUNIT Nickel
CsgA: THE MAIN CURLI SUBUNIT Nickel CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Nickel Curli x1 CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Nickel CsgA x n Curli x1 CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Bacteria Nickel CsgA x n Curli x1 CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Bacteria Curli x n Nickel CsgA x n Curli x1 CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Bacteria Curli x n Nickel CsgA x n Curli x1 CsgA x1
CsgA: THE MAIN CURLI SUBUNIT Bacteria Curli x n Nickel CsgA x n Curli x1 CsgA x1
HisTag: TWO POSSIBLE CONFORMATIONS His CsgA
HisTag: TWO POSSIBLE CONFORMATIONS CsgA His CsgA His
MODELING HYPOTHESIS Curli polymerization His 1 Polymerized curli
MODELING HYPOTHESIS With His Tag Curli polymerization His 1 His 1 Ni Ni Ni Ni CsgA Polymerized curli
MODELING HYPOTHESIS With His Tag Curli polymerization … until His n His 1 His 1 His 3 Ni Ni Ni Ni Ni Ni His 2 Ni Ni Ni Ni Ni Ni Ni His 1 Ni Ni Ni CsgA Polymerized curli CsgA
OUR BIOBRICKS TM BBa_K1404006 RBS CsgA No TAG CsgA
OUR BIOBRICKS TM BBa_K1404006 BBa_K1404007 RBS RBS CsgA His1 CsgA His 1 No TAG CsgA CsgA
OUR BIOBRICKS TM BBa_K1404006 BBa_K1404007 BBa_K1404008 RBS RBS CsgA RBS His1 CsgA CsgA His1 His2 His 1 No TAG His 2 His 1 CsgA CsgA CsgA
CURLI STRUCTURE NEGATIVE CONTROL NO CURLI
CURLI STRUCTURE NEGATIVE CONTROL POSITIVE CONTROL NO CURLI E.coli + BBa_K1404006 (No His Tag)
CURLI STRUCTURE CURLI FORMATION NEGATIVE CONTROL POSITIVE CONTROL NO CURLI E.coli + BBa_K1404006 (No His Tag)
CURLI STRUCTURE POSITIVE CONTROL E.coli + BBa_K1404007 E.coli + BBa_K1404006 (1 His Tag) (No His Tag)
CURLI STRUCTURE STRONG CURLI FORMATION POSITIVE CONTROL E.coli + BBa_K1404007 E.coli + BBa_K1404006 (1 His Tag) (No His Tag)
CURLI STRUCTURE STRONG CURLI FORMATION POSITIVE CONTROL E.coli + BBa_K1404007 E.coli + BBa_K1404008 E.coli + BBa_K1404006 (1 His Tag) (2 His Tags) (No His Tag)
CURLI STRUCTURE STRONG CURLI CURLI FORMATION FORMATION POSITIVE CONTROL E.coli + BBa_K1404007 E.coli + BBa_K1404008 E.coli + BBa_K1404006 (1 His Tag) (2 His Tags) (No His Tag)
BIOFILM STRUCTURE NEGATIVE CONTROL (CsgA- strain)
BIOFILM STRUCTURE E.coli + BBa_K1404006 NEGATIVE CONTROL (CsgA- strain) (No His Tag)
BIOFILM STRUCTURE E.coli + BBa_K1404006 E.coli + BBa_K1404007 E.coli + BBa_K1404008 (No His Tag) (1 His Tag) (2 His Tags)
BIOFILM STRUCTURE E.coli + BBa_K1404006 E.coli + BBa_K1404007 E.coli + BBa_K1404008 (No His Tag) (1 His Tag) (2 His Tags)
ADHERENCE Percentage of Adherence 100 90 80 70 Adherence (%) 60 50 40 30 20 10 0 CsgA- (negative No Tag (positive His 1 His 2 control) control) Crystal Violet Coloration
ADHERENCE Percentage of Adherence 100 90 80 70 Adherence (%) 60 50 40 30 20 10 0 CsgA- (negative No Tag (positive His 1 His 2 control) control) Crystal Violet Coloration
BIOFILM MATRIX Ni Ni Ni Ni Ni Ni Ni Ni Ni Ni
NICKEL CHELATION: PROCEDURE E.coli liquid culture 48 hours E.coli biofilm
NICKEL CHELATION: PROCEDURE E.coli liquid culture 48 hours E.coli biofilm Add Ni 2+
NICKEL CHELATION: PROCEDURE E.coli liquid culture 48 hours E.coli biofilm Add Ni 2+ Nickel chelation : 30 min E.coli biofilm containing Ni 2+
NICKEL CHELATION: PROCEDURE E.coli liquid culture 48 hours E.coli biofilm Add Ni 2+ Nickel chelation : 30 min Ni Concentration: E.coli biofilm containing Ni 2+ ICP – MS
NICKEL CHELATION: RESULTS Determination of Nickel chelated 0.9 0.8 0.7 µgNi / AU Curli 0.6 0.5 0.4 0.3 0.2 0.1 0 WT HIS 1 HIS 2
NICKEL CHELATION: RESULTS Determination of Nickel chelated 0.9 0.8 0.7 µgNi / AU Curli 0.6 0.5 0.4 0.3 0.2 0.1 0 WT HIS 1 HIS 2
NICKEL CHELATION: RESULTS Determination of Nickel chelated 0.9 0.8 0.7 µgNi / AU Curli 0.6 0.5 0.4 0.3 0.2 0.1 0 WT HIS 1 HIS 2
CsgA His 1
His 2 CsgA Ni Ni Ni Ni Ni Ni Ni His 1 Ni Ni Ni CsgA
OUR HP INSPIRED SOLUTION: FILTER CURLY’ON
OUR HP INSPIRED SOLUTION: FILTER CURLY’ON Polluted water
OUR HP INSPIRED SOLUTION: FILTER CURLY’ON Polluted water Purified water
OUR HP INSPIRED SOLUTION: FILTER CURLY’ON Polluted water Inside of the filter Purified water
OUR HP INSPIRED SOLUTION: FILTER CURLY’ON Polluted water Inside of the filter EXPERTS Damien Jacques, Design and Mechanical Engineering Baptiste Laubie University of Lorraine Expert in Recycling Purified water
HUMAN PRACTICE: sociological inquiry Inox industry Dialysis water depollution Food Recycling Processing
HUMAN PRACTICE: SURVEY WHICH PURIFICATION TECHNIC(S) DO YOU PREFER FOR DRINKING WATER ? 70 60 50 40 30 20 10 0 Chemical Method Physical method Biological method Alive bacteria Alive GM bacteria (proteins)
HUMAN PRACTICE: SURVEY WHICH PURIFICATION TECHNIC(S) DO YOU PREFER FOR DRINKING WATER ? 70 60 50 40 30 20 10 0 Chemical Method Physical method Biological method Alive bacteria Alive GM bacteria (proteins)
HUMAN PRACTICE: SURVEY Have you heard about synthetic biology? 70 60 PEOPLE SURVEYED (%) 50 40 30 20 10 0 NO YES
HUMAN PRACTICE: SURVEY WHICH PURIFICATION TECHNIC(S) DO YOU PREFER FOR DRINKING WATER ? 70 60 50 40 30 20 10 0 Chemical Method Physical method Biological method Alive bacteria Alive GM bacteria (proteins)
HUMAN PRACTICE: SURVEY WHICH PURIFICATION TECHNIC(S) DO YOU PREFER FOR DRINKING WATER ? 70 60 50 40 30 20 10 0 Chemical Method Physical method Biological method Alive bacteria Alive GM bacteria (proteins)
SAFETY: UV treatment No UV exposure After 10 min UV exposure After 15 min UV exposure After 20 min UV exposure
SAFETY: UV treatment No UV exposure After 10 min UV exposure Healthy bacteria Altered bacteria After 15 min UV exposure After 20 min UV exposure Dead bacteria
SAFETY: our complete solution UV Heating Ethanol Death of bacteria Degradation of genomic DNA Death of bacteria
SAFETY: our complete solution UV Heating Ethanol Death of bacteria Degradation of genomic DNA Death of bacteria
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