SLIDE 1
Crust Away
iGEM Trento 2012
HI!
UGL Y
Crust Away iGEM Trento 2012 Y UGL L L I T S Y L G U E. - - PowerPoint PPT Presentation
Crust Away iGEM Trento 2012 Y UGL L L I T S Y L G U E. - - PowerPoint PPT Presentation
HI! Crust Away iGEM Trento 2012 Y UGL L L I T S Y L G U E. coli Could bacteria provide a solution? Bad! Black Crust In the air Acid Rain Pollutants CaSO 4 H 2 SO 4 CaCO 3 H 2 SO 4 CaCO 3 SO 2 + H 2 O H 2 SO 3 Marble Surface
SLIDE 2
SLIDE 3
S T I L L U G L Y
SLIDE 4
Could bacteria provide a solution?
- E. coli
SLIDE 5
iGEM T rento 2012 Jason Fontana
Marble Surface
CaCO3
In the air
SO2 + H2O
Acid Rain
H2SO4
Black Crust
H2SO3
Bad!
CaCO3
CaSO4
Pollutants
H2SO4
SLIDE 6
Strictly Anaerobic Not controllable
Not genetically tractable
SRB
SLIDE 7
iGEM T rento 2012 Jason Fontana
Aerobic Not harming the marble Safe Controllable Cost efficient
Alternative Sulfate Reducing Pathway
Solution
SLIDE 8
S U L F A T E
OUT IN
CysDes
CYSTEINE
H
2
S CysE
M256I
SERINE
SLIDE 9
BLACK CRUST
OUT IN
CysDes
CYSTEINE
CysE
M256I
SERINE GAS
SLIDE 10
OUT IN
LacI-lacIq CysDes
RBS
Ptac lacO CYSTEINE
Arabinose IPTG MARBLE
B B a _ K 7 3 1 3 B B a _ K 7 3 1 4 araC-pBAD
CysE
RBS
GAS BLACK CRUST M256I
SLIDE 11
sfGFP
araC-pBAD
M256I CysE
RBS
Expression
110 220 330 440 1 2 3 4 5 6
Intensity of fluorescence (a.u.)
Time (h) + GLYCEROL + GLUCOSE
SLIDE 12
OUT IN CYSTEINE
Arabinose
B B a _ K 7 3 1 3 araC-pBAD
CysE
RBS
M256I
SULF A TE
+ Ninhydrin
Ruhemann’s Purple
SLIDE 13
0.0125 0.0250 0.0375 0.0500
[Cys] (mM)
araC-pBAD
M256I CysE
RBS
Carbon source Arabinose Glycerol
- Glycerol
+ Glucose
- Glucose
+ Empty Cells
Cysteine Production
SLIDE 14
OUT IN CYSTEINE
LacI-lacIq CysDes
RBS
Ptac lacO
IPTG
GAS
B B a _ K 7 3 1 4
SLIDE 15
sfGFP
LacI-lacIq CysDes
RBS
Ptac lacO
- Cysteine
+ Cysteine
125 250 375 500 0 hours 4 hours Intensity of fluorescence (a.u.)
SLIDE 16
LacI-lacIq CysDes
RBS
Ptac lacO
CysDes Cysteine
- +
+
- +
+
H2S Production
0.3 0.5 0.8 1.0
[H2S] (mM)
SLIDE 17
LacI-lacIq CysDes
RBS
Ptac lacO
H2S Quantification
CysDes H2S Empty Cells
SLIDE 18
SLIDE 19
SULF A TE BLACK CRUST
OUT IN
LacI-lacIq CysDes
RBS
Ptac lacO CYSTEINE
H
2
S
Arabinose IPTG MARBLE
B B a _ K 7 3 1 3 B B a _ K 7 3 1 4 araC-pBAD
CysE
RBS
M256I
SLIDE 20
LacI-lacIq CysDes
RBS
Ptac lacO
sfGFP
araC-pBAD
M256I CysE
RBS
50 100 150 200 1 2 3 4 5 6
CysE-sfGFP fluorescence (a.u.) time (h)
- IPTG
+IPTG
- IPTG
SLIDE 21
0.0125 0.0250 0.0375 0.0500
[Cys] (mM) CysE Induced? CysDes Induced? Y Y Y N N Y N N
araC-pBAD
M256I CysE
RBS
LacI-lacIq CysDes
RBS
Ptac lacO
Cysteine Degradation
SLIDE 22
0.0125 0.0250 0.0375 0.0500
[Cys] (mM) CysE Induced? CysDes Induced? Y Y Y N N Y N N
araC-pBAD
M256I CysE
RBS
LacI-lacIq CysDes
RBS
Ptac lacO
Cysteine Degradation
WORKS!
SLIDE 23
Cot ransformation Cysteine Production
2
Expression
1
H2S Production
3
Cysteine Degradation
4
Black crust removal
5
Application
- n statues
CysE CysE CysE
CysDes CysDes CysDes
CysE
CysDes
SLIDE 24
Denser 18 g/L agar Gel Jelly - MOPS 6 g/L with cells Three 12 h applications
SLIDE 25
Test Control
SLIDE 26
Clean Marble The Crustonator
Acid Rain Simulator
DIY Black Crust!
BBQ Ashes
SLIDE 27
Synthetic black crust is removed by our system.
Pre-application After third 12 h application SEM image pre t reatment SEM image after third 12 h application
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Optimize coexpression Optimize application method Have definitive negative controls T est on different stones and black crust types
Achievements F uture Directions
Built 30 Parts Submitted 21 Parts Characterized 11 Parts Improved 2 Parts Aerobic sulfate reduction pathway Reconstructed artificial black crust Developed a new bioremediation method to clean marble SRB Safety Book Outreach Activities
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THANK YOU!