iit delhi eco - 2015.igem.org2015.igem.org/files/presentation/iit_delhi.pdf · •proof –igem iit...

IIT DELHI

Eco.coli

INTRODUCTION

INTRODUCTIONPollutants Emission Standards Annual Pollution

Emitted

Hydrocarbons 0.95 Kg 35 Kg

Carbon monoxide 43 Kg 261 Kg

NOx 0.64 Kg 17.3 Kg

Carbon-dioxide 0.19 Kg 5,190 Kg

POLLUTION CRUSADER

PRESENT FUTURE

PURPOSE OF TEAM ìGEM IIT DELHI

POLLUTION CRUSADER - SCRDRAWBACKS

• 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

Components of motor exhaust

HEADSTART - IGEM 2014 PARTS SUBMITTED

NITRITE REDUCTASE SULPHIDE QUIONONE REDUCTASE

CHARATERIZATION

Nessler’s testFAILED

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


Methodology

Cloning Strategy

1. Cloning a Yellow Fluorescence Protein downstream

2. Cloning a strong promoter + Strong RBS upstream

NosZ


Not 3A assembly compatible

SYFP-2

SYFP-2

Methodology – CLONING STRATEGY

NosZ

Methodology - CLONINGCloning Strategy

SQR

Cloning a strong promoter + Strong RBS upstream


Results

Pollution Crusader-

Parts Submitted

Bba_K1866000

Bba_K1866004

Bba_K1866003Bba_K1866002

Bba_K1866001

P + RBS + NrfA

NosZ + YFPP + RBS + NosZ

P + RBS + SQRP + 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 & ResultsCharacterisation 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!



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




• 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




Luria Broth Clone containing nrfA

Nitrite (NO2-)

Growth

After 16 hours, for different nitrite conc.,

Monitor pHSUCCESSFUL

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




• 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 concentrations of nitrite (NO2

-) become toxic for the cells, due to which cell death occurs.

DH5 alpha (control) NrfA Clone

pH Monitoring




• 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 Indophenol Test

Reference-Koroleff, F. 1976. Determination of ammonia. In Methods of Seawater Analysis (K. Grasshoft, ed.). Verlag Chemie




Nitrite (NO2-)

Growth


After different time intervals, for Nitrite concentration = 1mM,

1ml culture aliquot

Indophenol test

Check OD 540SUCCESSFUL




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




Nitrite (NO2-)

Growth


1ml culture aliquot

Indophenol test

Check OD 540

SUCCESSFUL





• 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.




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.





Nitrite (NO2-)

Growth


1ml culture aliquot

Nessler’s reagent test CentrifugePour out media and weigh

SUCCESSFUL




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 GERMANYIGEM 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

Complete assembly

POLLUTION CRUSADER-prototype components

Frugal diesel burner to compensate for unavailability of engine

Tube containing silica gel

POLLUTION CRUSADER- working prototype

Ice-cold water for heat exchanger

De-sooter tankSuction 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 AWARNESSSOCIAL AWARNESS – GROUND LEVEL WORK

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

iit delhi eco - 2015.igem.org2015.igem.org/files/presentation/iit_delhi.pdf · •proof –igem iit...

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