iGEM

The iGEM Foundation is dedicated to education and competition, advancement of synthetic biology, and the development of open community and collaboration.

The main program at the iGEM Foundation is the International Genetically Engineered Machine (iGEM) Competition.

It is the premiere student competition in Synthetic Biology. Since 2004, participants of the competition have experienced education, teamwork, sharing, and more in a unique competition setting.

iGEM IIT Delhi

IIT Delhi has been participating in the competition for the past two years (iGEM 2013 and iGEM Giant Jamboree 2014).

Won a Bronze medal in 2014.

Project 2014 : Eco Coli

Greenhouse gases such as NOx and SOx pose a major global issue.

Our team planned to reduce the amount of the NOx and SOx gases ejected through the exhaust vents.

We genetically engineered E.coli and equipped it with the genes to synthesize

nitrite reductase enzyme (NrfA gene) : reduces NOx to NH3

sulfite reductase enzyme (CysI gene) : reduce SO2 to H2S

Sulfide Quinone reductase enzymes (Sqr gene) : reduces H2S to S.

These would be immobilized on polymer beads that have a positive zeta potential and placed in a bioreactor.

The bacteria will reduce oxides present in the incoming gas stream and consequently the percentage of NOx and SOx in the outgoing gas stream will be significantly lower.

Motivation for the project

Air pollution is of prime concern in today’s day and age.

Diesel generators produce a large amount of NOx and SOx , along with CO and CO2

Current methods for pollution reduction from diesel generators involve SRCs, which can provide high efficiency of pollution control only if the operating temperature is low, which in turn compromises with the efficiency of the machine.

Therefore, if a solution to this problem can be found biologically, it would solve this particular problem.

Some Pollution Stats

Component Emission Rate Annual Pollution Emitted

Hydrocarbons 1.75 g/Km 35 Kg

Carbon monoxide 13.06 g/Km 261 Kg

NOx 0.87 g/Km 17.3 Kg

Carbon-dioxide 258 g/Km 5,190 Kg

Component Emission Rate Annual Pollution Emitted

NMOG(Volatile organic compound) 0.046 g/Km 0.95 Kg

Carbon monoxide 2.1 g/Km 43 Kg

NOx 0.0305 g/Km 0.64 Kg

Formaldehyde 0.0092 g/Km 0.19 Kg

U.S. Environmental Protection Agency estimates of average passenger car emissions in the United States for July 2000:

United States Light-Duty Vehicle, Light-Duty Truck, and Medium-Duty Passenger Vehicle—Tier 2 Exhaust Emission Standards

Work done in iGEM 2014

We cloned the NrfA gene and Sqr gene under a constitutive promoter completely and got it’s sequencing done.

But due to shortage of time and resources, we could not complete the cloning of the CysI gene

We were awarded a bronze medal for our work, at MIT, in the giant Jamboree 2014.

Goals for iGEM 2015

We are determined to win a gold this year, and have therefore been brainstorming for ideas right from the start.

It was decided that we will be working on 2 projects this year:

Last year’s project (NOx and SOx reduction)

The other project would be finalized after a few sessions of brainstorming and discussion. Possible ideas:

Electricity production using Geobacter

The Biological camera

Biological Method for Ulcer Detection

Spore formation in Bacillus subtilis

Production of ethyl pyruvate

Working Plan for iGEM 2015

Project 2014 will be extended:

The genetically engineered bacteria will be immobilized onto reactors, and will be used to reduce the pollution from diesel generators.

The project this year will include cloning of genes reducing N2O to N2 (nosZ gene, Nitrous oxide reductase)

Reactor Flow Chart

Diesel generator

Muffler to reduce pressure waves

Heat exchanger

Spiral/honey comb reactorCoated with E.COLI

Nutrient injector

Final Exhaust

Compare NOx

Bubbling through water to generate tar

Working

1. Engine would be modified to work at higher temperatures, this would decrease the soot and CO emissions and increase NOx, which is desirable for us.

2. Soot would further be filtered out in water, forming tar which can be separated from the system, this ensure clog-free running of our system. Also, increasing moisture in exhaust, which is good for bacteria.

3. Exhaust goes to heat exchanger where temperature of the exhaust is brought down to 35C

4. Exhaust now goes to baffles where we achieve pressure drop and pressure regularization.

5. Finally exhaust is passed through our reactor, which is continuously supplied with nutrients through a mist sprayer.

6. Finally the exhaust is checked and values compared with initial values to calculate efficiencies.

The Complete Model

NOxRemoved by E.Coli

SOxNegligible quantities in Indian fuels

CORemoved by operating engine at higher temps and DOC

Soot and Tar

Filtered by bubbling through water and treating the leftover concentrated tar

Benzene IF something could be done practically/virtually, it could be of huge benefit since benzene above 5 ppm is carcinogenic

CO2 If we use some autotrophic bacteria, it would be the only technology that caters to CO2 emissions as well

Design Considerations and Alternatives

1. Engine operating temperature can be adjusted by altering coolant mass flow rates.

2. Nutrient injectors are hacked diesel injectors(Rs.200-400)

3. Heat exchangers are modified hacked car radiators. Further temp reduction would be achieved by evaporative cooling.

4. Main reactors would be modular in design such that different modules could be coated with different bacteria as per requirement and stacked up in desired sequence.

Critical points to work on:

1. Parameters that we can adjust to control NOx (like something to boost E.Coli population/ making them more active/ controlling nutrient levels)

2. How to sense NOx concentrations and compare initial and final levels (some sensor or anything)

3. Get quantitative data of everything cross checked.

4. Arrange for exhaust, reactor and NOx sensor and start experimenting to derive relation between surface area and NOx % reduction.(If we can get it directly, its good..otherwise we will have to experiment and use matlab to find relation)

5. Work on tar decomposing bacteria( papers of team that was disintegrating petroleum spillage)

6. Finding autotrophic bacteria.

7. Finding bacteria for decomposing benzene rings.

Some Good ideas for the second project from brainstorming sessions

The Biological camera

In order to take a picture, the researchers created a lawn of bacteria on a plate coated with a sugar called S-gal, which doubles as a dye.

When the lawn is exposed to a laser through an image mask, it releases an enzyme, LacZ, that reacts with the sugar, creating a black pixel under each E. coli.

We planned to extend this idea by cloning 3 pigment producing genes (coding for red, green and blue pigments), And thereafter exposing the image thrice, and superimposing the image thus formed.

Electricity production using Geobacter

To use Geobacter sulfurreducens to form a Microbial fuel cell with Sodium Acetate as the electron donor and a graphite electrode as the terminal electron acceptor.

Increasing the expression of gene responsible for extracellular electron transfer (Porin cytochrome complex) would lead to higher potential generation.

Use generated potential from here for regular electrical applications (e.g.- lighting LEDs on a small scale)

The second project The second idea that has been decided is to curb mosquitoes biting

humans:

Mosquitoes have CpA neurons, which help them sense CO2, thereby enabling them to find and bite us, even in the absence of light.

To this effect, we will be producing Ethyl Pyruvate using synthetic biology. Ethyl pyruvate is known to block the CpA neuron in Mosquitoes.

Ethyl pyruvate formation

Ethyl Pyruvate is an ester of Ethanol and Pyruvate (Pyruvic acid).

We will have two separate reactors, one in which Ethanol would be produced, and another in which Pyruvate would be produced (both from bacteria)

Since ethanol is the end product in anaerobic fermentation, it won’t be hard to produce.

Therefore, our main focus is on the production of Pyruvate Bacterially.

Pyruvate Production Some enzymes are known to catalyse the production of pyruvate in E.coli, when the right

substrate is provided, e.g.-

 a)  Enzymes such as formaldehyde dehydrogenase, pyruvate synthase, glycolate oxidase, tartrate dehydratase, and D-amino acid oxidase , have been employed in the enzymatic synthesis of pyruvate.

b) Pyruvic acid can be produced from Acetaldehyde and CO2 using PDC(pyruvate dehydrogenase complex)

c) Spinach GO (glycolate oxidase) and catalase( degrade H2O2 produced during action of GO on l- lactate) expressed in  Pichia pastoris can used as a whole-cell biocatalyst for pyruvate production. Can convert l-lactate (cheap substrate) into pyruvate.

d) lactate oxidase (LOX)-component-producing strains, namely Edwardsiella sp. and Pseudomonas sp. can effectively oxidize D, L-lactate, and racemic lactate into

pyruvate without hydrogen peroxide production. LOX catalyzes the reaction as follows.

CH3-CH(OH)-COOH+0.5 O2→CH3-CO-COOH+H2

e) NAD-independent lactate dehydrogenases( iLDHs )catalyze the oxidation of lactate to pyruvate in a flavin-dependent manner (flavin mononucleotide and flavin adenine dinucleotide for L- and D-iLDH, respectively). Pseudomonas stutzeriSDM, which has a good ability to produce pyruvate from lactate, has been reported by Hao et al. 

Working Plan for Ethyl Pyruvate Production

Look for the best enzyme (among those listed in the previous slide). This means the enzyme with the best conversion rate, high efficiency, ease of access.

Look for the gene sequence and clone the gene from the original organism into E.coli

Alternatively, look for biobricks submitted by other teams related to this, in the iGEM registry and work on improving those.