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 iGEM T rento 2012 Jason Fontana

Strictly Anaerobic Not genetically tractable SRB Not controllable

Aerobic Not harming the marble Solution Alternative Sulfate Reducing Pathway Safe Controllable Cost efficient iGEM T rento 2012 Jason Fontana

E T A F L U S SERINE OUT IN CysE M256I CYSTEINE CysDes S H 2

BLACK CRUST SERINE OUT IN CysE M256I CYSTEINE CysDes GAS

MARBLE BLACK CRUST Arabinose RBS araC-pBAD CysE M256I B B a _ K 7 3 1 0 3 0 OUT IN CYSTEINE RBS CysDes LacI-lacIq P tac lacO B B a _ K 7 3 1 4 0 0 IPTG GAS

440 Intensity of fluorescence (a.u.) 330 220 110 0 0 1 2 3 4 5 6 Time (h) + GLYCEROL + GLUCOSE Expression RBS araC-pBAD M256I CysE sfGFP

TE A SULF Arabinose RBS araC-pBAD CysE B B a _ K 7 3 1 0 3 0 M256I OUT IN CYSTEINE Ruhemann’s + Ninhydrin Purple

Carbon source Glycerol Glycerol Glucose Glucose Empty Cells Arabinose - + - + 0.0500 [Cys] (mM) 0.0375 0.0250 0.0125 0 Cysteine Production RBS araC-pBAD M256I CysE

OUT IN CYSTEINE RBS CysDes LacI-lacIq P tac lacO B B a _ K 7 3 1 4 0 0 IPTG GAS

500 Intensity of fluorescence (a.u.) 375 250 125 0 0 hours 4 hours - Cysteine + Cysteine RBS CysDes LacI-lacIq sfGFP P tac lacO

+ - + - CysDes + - - + Cysteine 1.0 [H2S] (mM) 0.8 0.5 0.3 0 H 2 S Production RBS CysDes LacI-lacIq P tac lacO

CysDes H 2 S Empty Cells H 2 S Quantification RBS CysDes LacI-lacIq P tac lacO

MARBLE BLACK TE A SULF CRUST Arabinose RBS araC-pBAD CysE B B a _ K 7 3 1 0 3 0 M256I OUT IN CYSTEINE RBS CysDes LacI-lacIq P tac lacO B B a _ K 7 3 1 4 0 0 IPTG S H 2

CysE-sfGFP fluorescence (a.u.) 200 150 100 50 0 0 1 2 3 4 5 6 time (h) -IPTG +IPTG -IPTG RBS sfGFP araC-pBAD M256I CysE RBS CysDes LacI-lacIq P tac lacO

Cysteine Degradation CysE Induced? Y Y N N CysDes Induced? Y N Y N 0.0500 [Cys] (mM) 0.0375 0.0250 0.0125 0 RBS CysDes RBS LacI-lacIq araC-pBAD M256I CysE P tac lacO

Cysteine Degradation WORKS! CysE Induced? Y Y N N CysDes Induced? Y N Y N 0.0500 [Cys] (mM) 0.0375 0.0250 0.0125 0 RBS CysDes RBS LacI-lacIq araC-pBAD M256I CysE P tac lacO

CysE CysDes CysE CysDes Black crust CysDes Cysteine H 2 S Expression CysDes removal Degradation Production CysE 1 2 4 3 5 Cot ransformation Application on statues Cysteine Production CysE

Denser 18 g/L agar Gel Three 12 h applications Jelly - MOPS 6 g/L with cells

Test Control

BBQ Ashes Clean Marble The Crustonator DIY Black Crust! Acid Rain Simulator

Pre-application After third 12 h application SEM image after third 12 h application SEM image pre t reatment Synthetic black crust is removed by our system.

Achievements F uture Directions Built 30 Parts Optimize coexpression Submitted 21 Parts Optimize application method Characterized 11 Parts Have definitive negative controls Improved 2 Parts T est on different stones and Aerobic sulfate reduction pathway black crust types Reconstructed artificial black crust Developed a new bioremediation method to clean marble SRB Safety Book Outreach Activities

THANK YOU!