IIT DELHI Eco.coli INTRODUCTION INTRODUCTION Poll ollutants Em - - PowerPoint PPT Presentation

IIT DELHI Eco.coli

INTRODUCTION

INTRODUCTION

Poll

• llutants

Em Emis issio ion St Standards Annual l Poll

• llution

Em Emit itted Hy Hydrocarbons 0.95 Kg 35 Kg Car Carbon mon

• noxide

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

• f
• f 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 3) CYTOCROME C HEMOPROTEIN 4) SOOT / RSPM

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

Nitrite Reductase (nrfA)

Our “Part Bs”

From E.coli

From P.aeruginosa

Sulfite reductase (cysI) Nitrous oxide reductase (nosZ)

From Synechococcus

Sulfide quinone Reductase (SQR)

NO/NO2

• NH3

N2O N2 SO2 H2S H2S S

Methodology – CLONING STRATEGY

Cloning Strategy

• 1. Cloning a strong promoter + Strong RBS upstream
• 2. Cloning a Yellow Fluorescence Protein downstream

NrfA SYFP-2

3A assembly compatible 3A assembly compatible

Methodology

Cloning Strategy

• 1. Cloning a Yellow Fluorescence Protein downstream
• 2. Cloning a strong promoter + Strong RBS upstream

NosZ 3A assembly compatible Not 3A assembly compatible SYFP-2 SYFP-2

Methodology – CLONING STRATEGY

NosZ

Methodology - CLONING

Cloning Strategy

SQR

Cloning a strong promoter + Strong RBS upstream

3A assembly compatible

Results

Pollution Crusader- Parts Submitted

Bba_K1866000 Bba_K1866004 Bba_K1866003 Bba_K1866002 Bba_K1866001 P + RBS + NrfA NosZ + YFP P + RBS + NosZ P + RBS + SQR P + RBS + NrfA +YFP +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

Nitrate Fumarate Luria Broth(5%)

Minimal Media Clone containing nrfA

Nitrite (NO2

• )

FAILED

No growth

Minimal Media Growth

Reference- Clarke, TA., Mills, PC. et al (2008). Escherichia coli cytochrome c nitrite reductase NItalic textrfA.. Methods in Enzymology

Characterisation & Results

Characterisation of Part Bba_K1866000

Promoter + RBS + NrfA Functional assay of Protein

• 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 Minimal Media Growth

Characterisation & Results

Characterisation of Part Bba_K1866000

Promoter + RBS + NrfA Functional assay of Protein

Luria Broth Clone containing nrfA

Nitrite (NO2

• )

Growth

After 16 hours, for different nitrite conc.,

Monitor pH

SUCCESSFUL

pH Monitoring

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

• The experiment showed results as expected, with

the pH of our clones being higher than the control, which can be attributed to the presence

• f 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

concentrations of nitrite (NO2

• ) become toxic for

the cells, due to which cell death occurs.

DH5 alpha (control) NrfA Clone

pH Monitoring

Characterisation & Results

Characterisation of Part Bba_K1866000

Promoter + RBS + NrfA Functional assay of Protein

• Cultures of our clones were grown anaerobically in 5ml Luria

broth, along with standard DH5α 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 The e In Indophenol l Tes est

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 (NO2

• )

Growth

Luria Broth

Clone containing nrfA

After different time intervals, for Nitrite concentration = 1mM, 1ml culture aliquot

Indophenol test

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 (NO2

• )

Growth

Luria Broth Clone containing nrfA 1ml culture aliquot

Indophenol test

Check OD 540

SUCCESSFUL

After 16 hours, for different nitrite conc.,

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

Luria Broth Clone containing nrfA

Nitrite (NO2

• )

Growth

After 16 hours, for different nitrite conc.,

1ml culture aliquot

Nessler’s reagent test Centrifuge

Pour out media and weigh

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 BIELEFELD GERMANY IGEM TEAM MACQUARIE Many teams working on human hemoglobin iGEM14 TU Delft-Leiden

HEME PROBLEM – WHAT EXACTLY IS THE PROBLEM?

3) TRANSPORTAION OF HEME TO PERIPLAMIC SPACE 1)HEME PATHWAY IS HIGHYLY REGULATED 4) MATURATION OF HEME BY CcmAH COMPLEX IS DONE. 2) Fe+2 INCORPORATION REQUIRED 5) DOCKING OF ENZYME WITH HEME.

HEME – MODELLING IN COPASI

COPASI

• PARAMETERS WERE

TAKEN FROM – BRENDA,KEGG, ECOCYC

• LITERATURE

SCANVING OF HEME PATHWAYS

Methodology

COPASI

PROBLEM MIGHT THEIR AT THE DOWNSTREAM OF TRANSPORTAION PROCESS

• AT HEME

MATURATION.

OVEREXPRESSING CcmAH COMPLES

• RATE LIMITING STEP IN

DOCKING

• COMPLEX CASSETE IS VERY

LARGE

• YAC / BAC SHOULD BE USED

TO CLONE THE CASSETE.

SOLUTIONS TO HEME PROBLEM

EXTRACELLULAR 1) Incorporation of ChuA receptor. 2)Provide ALA Extracellularly.

INTRACELLAR 1) Clone ALA synthase of rat to avoid feedback inhibition by heme. 2)Overexpression of CcmAH complex.

POLLUTION CRUSADER – MECHANICAL PART

POLLUTION CRUSADER-prototype

Exhaust from engine De-sooter tank removes Hydrocarbons Blower pushes gases through silica gel where NOx gets converted to NO NO rich gases passed through culture containing Eco.coli Com Complete asse assembly

POLLUTION CRUSADER-prototype components

Fru Frugal l die diesel l bu burner to to com

• mpensate for
• r un

unavaila lability

• f
• f eng

engine Tube containin ing si sili lica gel el

POLLUTION CRUSADER- working prototype

Ic Ice-cold wat ater for

• r

he heat exchanger De De-so sooter tank Su Suction pu pump

POLLUTION CRUSADER – RESULTS

The pollution crusader prototype showed a positive result, with the desooter tank working even better than expected. Heat exchanger was efficient in reducing the temperature substantially, which was monitored by a gas thermometer. Subsequently, NOx sensors placed after the silica gel pipe showed that the silica gel also worked as expected, reducing NO2 to NO. The NOx sensor showed negligible amounts of NO2 coming out of the pipe.

POLLUTION CRUSADER – RESULTS

Run number amount of soot collected(gram) 1 23.5 2 34.6 3 26.7 4 19.9 5 24.1 Run number NO2 concentration (standard, ppm) NO2 conc (detected) 1 42 9 2 42 8 3 42 11 4 42 9 5 42 10

CONCLUSIONS

1) CLONING OF ALL THE PARTS WERE SUCCESSFUL. 2) NRFA IS WORKING AS EXPECTED. 3) NRFA WAS CHARACTERIZED BY FOUR DIFFERENT ASSAYS. 4) 3 SHOWED ENZYME IS WORKING AND 1 ASSAY FAILED 5) PROTOTYPE IS WORKING AS EXPECTED.

ACHIEVEMENTS

1) 5 BIOBRICKS SUBMITTED. 2) 3 ARE STANDARD BIOBRICKS. 3) A PART HAS BEEN EXTENSIVELY CHARACTERIZED. 4) PROTOTYPE IS WORKING. 5) TWO EXISTING PART HAS BEEN IMPROVED BY PLACING SYFP2 DOWNSTREAM OF GENE

HUMAN PRACTICES

GOVERNMENT – POLICY CHANGES

DELHI CHIEF MINISTER

• CONCERNS OVER

ENVIRONMENTAL EXPOSURE

• CONVINCED
• SUPPORTS, FUND OUR

PROJECT

• PROOF – IGEM IIT DELHI 2016 –

DELHI GOVT SPONSORED

• BEAURACRATIC LEVEL STUCK –

2015

SOCIOLOGIST RESEARCH

PhD STUDY

• ETHICAL STUDY
• STUDIED - TEAM WORK
• RESEEARCH WILL COME

OUT SOON

• HUMANITARIAN MENTOR

OF THE TEAM

ORIENTATION – BEST BIOTECHNOLOGY COLLEGES

SYNBIO INITIATION

• 20 COLLEGES

PARTICIPATED

• MORE THAN 1000

STUDENTS

• TWO COLLEGES(NSIT

& DTU) WILL REGISTER FOR IGEM - 2016

SOCIAL AWARNESS – GROUND LEVEL WORK

SOCIAL AWARNESS SOCIAL AWARNESS – GROUND LEVEL WORK

SOCIAL AWARNESS

SOCIAL AWARNESS

SOCIAL AWARNESS

TRYST – OPEN HOUSE

1)Tryst is India’s biggest annual science and technology festival. 2) iGEM Team IIT Delhi was awarded the 1st runner up prize in the ‘Best stall in Tryst 2015’ category  WON $302. 3)Open house is an annual event in IIT Delhi which aims to promote and increase awareness about new technologies specifically for school children. 4) iGEM team IIT Delhi put up a stall in this event with the goal to Motivate and encourage school students to develop a greater interest in Biology and Biotechnology. 5) We also gave a basic introduction about synthetic biology and iGEM with the hope that it might motivate some of them to enrol as the first high school team to participate in iGEM from India.

OTHER HUMAN PRACTICES WORK

1) COMPILED POLLUTION DOUCMENT. IT IS 179 PAGE BOOK ON POLLUTION IN INDIA. MOST EXTENSIVE STUDY OF AIR POLLUTION IN INDIA. (PDF LINK ON WIKI) 2) SUBMITTED THE REPORT TO ENVIORNMRNT AND HEALTH MINISTRY, GOVT. OF INDIA. 3)HELPED IIT KHARAGPUR, IIT MADRAS. 4)ATTENDED IGEM INDIA MEET AT IISER.

SURVEY

1) iGEM Team IIT Delhi conducted some surveys to get an idea of the level of awareness among the citizens of Delhi 2) This was done in Central Park in Connaught Place, New Delhi and Munerika village. 3) The details of the surveys have been enclosed in a separate document.

CROWDFUNDING

ROCKY ROAD

OUR SPONSORS