IIT DELHI Eco.coli
INTRODUCTION
INTRODUCTION Poll ollutants Em Emis issio ion St Standards Annual l Poll ollution Em Emit itted Hydrocarbons Hy 0.95 Kg 35 Kg Car Carbon mon onoxide 43 Kg 261 Kg NOx 0.64 Kg 17.3 Kg Car Carbon-dioxide 0.19 Kg 5,190 Kg
POLLUTION CRUSADER PRESENT FUTURE
PURPOSE OF TEAM ìGEM IIT DELHI
POLLUTION CRUSADER - SCR DRAWBACKS • Expensive Pt and Pd metals used. • Over time performance deteriorates; entire setup has to be replaced/replenished. • Non recyclable; adds to junk on earth. Non biodegradable. • Needs high temp to work properly.
POLLUTION CRUSADER - RSPM
Eco . coli POLLUTION CRUSADER
POLLUTION CRUSADER – JOURNEY BEGINS
INTRODUCTION Co Componen ents ts of of motor exh xhaust
HEADSTART - IGEM 2014 PARTS SUBMITTED NITRITE REDUCTASE SULPHIDE QUIONONE REDUCTASE
CHARATERIZATION Nessler’s test FAILED
TROUBLESHOOTING
PROBLEMS – TURNING POINT 1) NO RBS UDOWNSTREAM OF PROMOTER 2) PERIPLASMIC PROTEIN 4) SOOT / RSPM 3) CYTOCROME C HEMOPROTEIN
POLLUTION CRUSADER SOOT / RSPM • PM10 highly dangerous • Wears down respiratory tract, exposing to NOx and SOx • 47% in air • Doctor’s advice – No workout/exercise
SOOT / RSPM SOLUTION MECHANICAL SYSTEM PROTOTYPE TEAM WAS FORMED RIGHT AWAY LOOKING AT MECHANICAL ASPECT OF PROJECT.
POLLUTION CRUSADER – BIOLOGICAL PART
BIOLOGICAL PART - CLONING 1) NO RBS UDOWNSTREAM OF PROMOTER 2) PERIPLASMIC PROTEIN
PERIPLASMIC PROBLEM Two of our genes (nrfA and nosZ) coded for periplasmic . proteins Solution- Doing a protein blast on the genes helped us find that they contain a hydrophobic alpha helix, which will guide the protein to the periplasmic space
Methodology How we cloned, what we cloned! Part A = Constitutive promoter + Strong RBS Part B = Pollution Crusader Gene
Methodology Our “Part Bs ” Nitrite Reductase (nrfA) NO/NO 2 NH 3 - From E.coli Nitrous oxide reductase (nosZ) N 2 O N 2 Sulfite reductase (cysI) SO 2 H 2 S From P.aeruginosa Sulfide quinone Reductase (SQR) H 2 S S From Synechococcus
Methodology – CLONING STRATEGY Cloning Strategy NrfA 1. Cloning a strong promoter + Strong RBS upstream 3A assembly compatible 2. Cloning a Yellow Fluorescence Protein downstream 3A assembly compatible SYFP-2
Methodology Cloning Strategy NosZ 1. Cloning a Yellow Fluorescence Protein downstream SYFP-2 Not 3A assembly compatible 2. Cloning a strong promoter + Strong RBS upstream 3A assembly compatible SYFP-2
Methodology – CLONING STRATEGY NosZ
Methodology - CLONING Cloning Strategy SQR Cloning a strong promoter + Strong RBS upstream 3A assembly compatible
Results Bba_K1866000 P + RBS + NrfA Bba_K1866004 Bba_K1866001 NosZ + YFP P + RBS + NosZ Pollution Crusader- +YFP Parts Submitted Bba_K1866002 Bba_K1866003 P + RBS + NrfA P + RBS + SQR +YFP
Characterisation & Results Clone Confirmation by Double Digestion • Lane 3- Bba_K1866000 • Lane 4- Bba_K1866002 • Lane 6- Bba_K1866001 • Lane 8- Bba_K1866003
Characterisation & Results Clone Confirmation by Sequencing • Plasmids from our clones were extracted and sent to eurofins for sequencing • Sequencing revealed that the cloning that we had done was correct
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Lane 6- Total protein content for clone containing NrfA Lanes 3&4- Periplasmic fractionation for clone containing NrfA Bands are exactly where we want them!
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein Minimal Media Growth Nitrate Fumarate Luria Broth(5%) Minimal Media - ) Nitrite (NO 2 Minimal Media No growth Clone containing nrfA FAILED Reference- Clarke, TA., Mills, PC. et al (2008). Escherichia coli cytochrome c nitrite reductase N Italic text rfA.. Methods in Enzymology
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein Minimal Media Growth • Neither the control (DH5 alpha), nor our clones showed growth on minimal media after 18 hours. • A white pellet was obtained, but later we realized that the pellet was of ampicillin, which had formed a white precipitate with formate
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein pH Monitoring - ) Nitrite (NO 2 Growth Luria Broth Clone containing nrfA After 16 hours, for different nitrite conc., SUCCESSFUL Monitor pH Reference- Evaluation of pH indicator-based colorimetric films for ammonia detection using optical waveguides-J. Courbata, D. Brianda, J. Damon-Lacostea, J. Wöllensteinb, N.F. de Rooij
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein pH Monitoring • The experiment showed results as expected, with the pH of our clones being higher than the control, which can be attributed to the presence of excess ammonia in the solution. • We also saw that at concentrations of Nitrite greater than 2mM, the pH showed a sharp decline. This could be due to the fact that high - ) become toxic for concentrations of nitrite (NO 2 the cells, due to which cell death occurs. NrfA Clone DH5 alpha (control)
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein The e In Indophenol l Tes est • Cultures of our clones were grown anaerobically in 5ml Luria broth, along with standard DH5 α cells, used as control. These were then subcultured into 50 ml LB containing tubes, also grown anaerobically, with different concentrations of Sodium Nitrite. • To a 1ml aliquot, 40µl phenol, 40µl sodium nitroprusside and 100µl of oxidising reagent (tri- sodium citrate + sodium hypochlorite) was added, giving an orange-yellow colour • The absorbance of this was measured at a wavelength of 540nm Reference-Koroleff, F. 1976. Determination of ammonia. In Methods of Seawater Analysis (K. Grasshoft, ed.). Verlag Chemie
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein - ) Nitrite (NO 2 Growth Luria Broth Clone containing nrfA After different time intervals, for Nitrite concentration = 1mM, Indophenol test 1ml culture aliquot Check OD 540 SUCCESSFUL
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein The experiment showed results as expected, with the absorbance value of our clones being higher than the control, which can be attributed to the presence of excess ammonia in the solution. The value increased with increase in time, before finally saturating after ~16 hours
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein - ) Nitrite (NO 2 Growth Luria Broth Clone containing nrfA After 16 hours, for different nitrite conc., Indophenol test 1ml culture aliquot Check OD 540 SUCCESSFUL
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein • This experiment was also successful. The graph showed an increase in OD Values with increasing nitrite concentrations. • At high values of nitrite (>2mM), the graph shows a sharp decline, which can once again be attributed to Nitrite toxicity.
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein The Nessler’s test • On addition of Nessler’s reagent to a solution containing ammonia, a reddish brown precipitate is formed • This precipitate can be centrifuged and weighed after pouring out the solution. The weight of the precipitate gives an estimate of the relative amount of ammonia present in the solution. Reference- Mackie and MacCartney,1989,Practical Medical Microbiology, Collee J.G.,Duguid J.p.,Fraser A.G.and Marmion B.p. (Eds.),13th ed., Churchill Livingstone, edinburgh.
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein - ) Nitrite (NO 2 Growth Luria Broth Clone containing nrfA After 16 hours, for different nitrite conc., Pour out media and Nessler’s reagent test Centrifuge weigh 1ml culture aliquot SUCCESSFUL
Characterisation & Results Characterisation of Part Bba_K1866000 Promoter + RBS + NrfA Functional assay of Protein This experiment was also successful, showing trends as expected. At high nitrite concentrations, the pellet size reduced drastically (the reason being toxicity at high nitrite concentration).
HEME PROBLEM – PREVIOUS WORK IGEM TEAM MACQUARIE IGEM BIELEFELD GERMANY iGEM14 TU Delft-Leiden Many teams working on human hemoglobin
HEME PROBLEM – WHAT EXACTLY IS THE PROBLEM? 1)HEME PATHWAY IS HIGHYLY 4) MATURATION OF HEME BY REGULATED CcmAH COMPLEX IS DONE. 2) Fe+2 INCORPORATION 5) DOCKING OF ENZYME WITH REQUIRED HEME. 3) TRANSPORTAION OF HEME TO PERIPLAMIC SPACE
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