Since our target was to reduce air pollution, we targeted genes that act on NOx and SOx gases. Research was done extensively and the following genes were found to be best fitting for our project-
The general cloning strategy for NrfA consists of the following steps.
Nitrous oxide is a potent greenhouse gas, whose atmospheric concentration has been increasing since the introduction of the Haber Bosch process led to the widespread use of nitrogenous fertilizers. One of the pathways to its destruction is reduction to molecular nitrogen by the enzyme nitrous oxide reductase found in denitrifying bacteria.
NosZ, a gene coding for nitrous oxide reductase, isolated from Pseudomonas aeruginosa PAO1, is known to convert N2O to harmless Nitrogen gas (N2). It has two copper centers, a binuclear CuA center, similar to the one found in cytochrome c oxidase, and the CuZ center, a unique tetranuclear copper center now known to possess either one or two sulfide bridges.
The reduction of nitrous oxide to molecular nitrogen requires two protons and two electrons, according to the equation N2O + 2H+ + 2e− → N2 + H2O, E°′ (pH 7.0) = 1.35 V; ΔG0′ = −339.5 kJ/mol
This is a challenging reaction to be catalyzed by a metalloenzyme, as nitrous oxide is not just kinetically inert to decomposition but also a poor transition metal ligand, due to its weak σ-donating and π-accepting properties. Although, there have been some reports of other enzymes, such as nitrogenase and a multicopper oxidase from Pyrobaculum aerophilum, able to catalyze this reaction in vitro, the only one, whose primary role is the reduction of nitrous oxide, is N2O reductase (NosZ)
The complete denitrification pathway is shown below-
Gene construction & cloning:
The general cloning strategy for P+RBS+NosZ+SYFP consists of the following steps.
III - SQR (Sulfide Quinone Reductase)-
The SQR gene codes for sulfide quinone reductase, an enzyme catalysing the conversion of H2S to elemental Sulfur(S). The initial plan was to clone this gene downstream of the CysI gene from Pseudomonas aeruginosa, which codes for sulfite reductase, catalysing the conversion of sulfite to hydrogen sulfide (H2S).
Sulfur dioxide is a gas. It is invisible and has a nasty, sharp smell. It reacts easily with other substances to form harmful compounds, such as sulfuric acid, sulfurous acid and sulfate particles.
About 99% of the sulfur dioxide in air comes from human sources. The main source of sulfur dioxide in the air is industrial activity that processes materials that contain sulfur, eg the generation of electricity from coal, oil or gas that contains sulfur. Some mineral ores also contain sulfur, and sulfur dioxide is released when they are processed. In addition, industrial activities that burn fossil fuels containing sulfur can be important sources of sulfur dioxide.
Sulfur dioxide is also present in motor vehicle emissions, as the result of fuel combustion. In the past, motor vehicle exhaust was an important, but not the main, source of sulfur dioxide in air. However, this is no longer the case.
Sulfur dioxide affects human health when it is breathed in. It irritates the nose, throat, and airways to cause coughing, wheezing, shortness of breath, or a tight feeling around the chest. The effects of sulfur dioxide are felt very quickly and most people would feel the worst symptoms in 10 or 15 minutes after breathing it in.
Those most at risk of developing problems if they are exposed to sulfur dioxide are people with asthma or similar conditions.
The CysI gene converts sulfur dioxide, SO2 to hydrogen sulfide, H2S. However, hydrogen sulfide in itself is a harmful gas. The U.S. Environmental Protection Agency (U.S. EPA) identifies the most serious hazardous waste sites in the nation. U.S. EPA then includes these sites on the National Priorities List (NPL) and targets them for federal clean-up activities. U.S. EPA has found hydrogen sulfide in at least 35 of the 1,689 current or former NPL sites. Hence, cloning the SQR gene is important, as it will convert the toxic waste gas hydrogen sulfide to sulfur, which is a solid product and can therefore be separated out with ease.
Gene construction & cloning:
The general cloning strategy for NrfA consists of the following steps.
Confirmation of the clones:
We performed a double digestion by EcoRI and PstI to release the cloned fragment from the recombinant vector. The digestion products were resolved on an Agarose Gel by Electrophoresis and the size of the fragment confirmed the correct clone.
Figure3: Clone confirmation
IV - The Problem:Heme-
The biggest problem which we encountered was that the heme synthesis was much less as compared to our overexpressed protein. Heme :
Heme (iron-protoporphyrin IX) prosthetic groups perform a wide range of functions in nature including
Types of Heme :
The most common types of heme are heme b and heme c. Heme b: It is iron-protoporphyrin IX that is bound noncovalently to protein Heme c: heme c has two covalent thioether bonds formed between Cys side chains and the heme vinyl groups at positions 2 and 4 . The stereochemistry of heme attachment is the same in all known examples of heme c, and the vinyl groups at positions 2 and 4 are attached to the N- and C-terminal Cys of the CXXCH motif, respectively.
Other, less common, derivatives of heme include heme d1, present in cytochrome cd1 nitrite reductase, heme a, found in cytochrome c oxidase, and the related heme o, found in some bacterial oxidases. From a chemical perspective, hemes b and c are very similar to each other and thus are not expected to display significant inherent differences in electronic structure or reduction potential. From a biosynthetic perspective, however, these types of heme are quite different; because heme c is synthesized from heme b, the use of heme c requires a greater investment from the organism. The heme which is found in NrfA is Heme c
Heme Biosynthesis Pathway :
Cytochrome c biogenesis:
Natural modiﬁcations to heme occur after the ferrous iron (Fe2) is inserted into the porphyrin by ferrochelatase, the last enzyme in heme biosynthesis
CCm complex : It is a complex of Transmembrane protein which helps in Heme maturation .From modelling we believe that by upregulating CCm complex of proteins we can increase heme incorporation into our proteins.
Technique to improve heme incorporation:
V - Sulphite Reductase(Sir)-
NADPH-sulfite reductase (SiR) of Escherichia coli catalyzes the reduction of sulfite to sulfide and is required for synthesis of L-cysteine from inorganic sulfate The native enzyme has a subunit structure a8B4 , where a8 is a flavoprotein (SiR-FP) containing both flavin adenine dinucleotide and flavin mononucleotide and P is a hemoprotein (SiR-HP) containing an Fe4S4 centre and a single molecule of siroheme Electron flow between these cofactors proceeds from NADPH to flavin adenine dinucleotide to flavin mononucleotide in the flavoprotein, then to a closely coupled Fe4S4 siroheme center in the hemoprotein, and finally from siroheme to sulphiteThe SiR-FP and SiR-HP components of SiR are encoded by cysJ and cysl, respectively. These genes are contiguous and together with cysH, the gene for 3'-phosphoadenosine 5'-phosphosulfate sulfotransferase, comprise an operon with the gene order promoter-cysJ-cysI-cysH cysJIH operon is part of the positively regulated cysteine regulon (15) and requires sulfur limitation CysB protein, and either O-acetyl-L-serine or N-acetyl-L-serine for expression SiR activity is also dependent on cysG, which encodes a uroporphyrinogen III methyltransferase necessary for the synthesis of siroheme This gene is located more than 10 min away from cysJIH on the chromosomal map (34) and is not tightly regulated as part of the cysteine regulatory
VI - Characterization-
Poly Acrylamide Gel Electrophoresis
1. To characterise the Promoter + RBS + nrfA biobrick (Bba_K1866000), we first ran the total protein content of our recombinant E.coli DH5α culture (into which our pSB1A3 plasmid containing the biobrick had been cloned) on an SDS-Polyacrylamide Gel. The result showed over expression in the range of 55 kDa, which is roughly equal to the size of our protein.
2. Periplasmic protein SDS-PAGE—following the over expression obtained from the Poly Acrylamide gel electrophoresis, we extracted the protein content from the periplasmic space using the periplasm fractionation protocol. This was run on another polyacrylamide gel, and we saw over expression on this gel as well, on the appropriate range.
3. Minimal media growth- Following a research paper by Clarke et al., a culture of our cloned E.coli DH5α culture was grown on a minimal media containing 5% Luria Broth (by volume), 40mM Potassium Nitrate, 20mM Fumarate and 0.4% glycerol for 16 hours, in anaerobic conditions. The cells were then pelleted by centrifugation and were then to be sub cultured. This experiment failed, even though we saw a pellet after centrifugation following growth. However, we later realised that the pellet was not of our cells, but of ampicillin, which had formed some sort of white precipitate with the media. Hence, this resulted in a failure.
4. pH Monitoring (LB Growth)- Cultures of our clones were grown anaerobically in 5ml of luria broth, along with standard DH5α cells, used as control. These were then subcultured into 50 ml cultures, also grown anaerobically, with different concentrations of Sodium Nitrite. After 16 hours of growth, the cultures were taken out and the pH of the solution was taken for all different concentrations of Nitrite. 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 (NO¬2-) become toxic for the cells, due to which cell death occurs.
5. Indophenol test (LB Growth)- Cultures of our clones were grown anaerobically in 5ml in luria broth, along with standard DH5α cells, used as control. These were then subcultured into 50 ml cultures, also grown anaerobically, with different concentrations of Sodium Nitrite. To a 1ml aliquot, 40 microlitre phenol, 40 micolitre sodium nitroprusside and 100 ul of oxidising reagent (tri- sodium citrate + sodium hypochlorite) was added, and absorbance was measured at 540 nm. These OD values were plotted against DH5α culture, used as control for the experiments.
a. Monitoring with time- For a concentration of 1mM Nitrite, multiple cultures were inoculated. After regular intervals of time, the indophenol test was carried out, and OD540 values were noted. This experiment was successful, as it showed an increase of OD values with increase in time, before finally saturating. The OD values were more than that of DH5α cultures throughout, pointing to the fact that more ammonia was being formed in our clone cultures.
Why the "Pollution Crusader Device"?
RSPM and NOx when combined, form a deadly combination which drastically affects human health.
RSPM when inhaled acts like an abrasive and wears the lining of respiratory tract exposing soft spots where NOx reacts and causes severe issues. So we decided to eliminate soot and NOx as the primary objective.
Analysis of soot:
Soot is of two types: hydrophilic as well as hydrophobic. Hydrophilic soot dissolves in water, whereas hydrophobic soot dissolves in organic solvents like alcohol. To get rid of both the hydrophilic and hydrophobic components, we decided to use a solution of acetic acid in water as the solvent. Acetic acid, being an organic solvent, dissolves the hydrophobic part, and water, dissolves the hydrophobic soot. Using acetic acid, however, offers an added advantage as well. Being an acid, it reduces the PH of the medium and hence decreases the solubility of acidic NOx and SOx, from the exhaust into this solvent. Thus, the tank containing this solvent dissolves soot and NOx, Sox gases move out with the exhaust. The exhaust out of this tank is now passed into the desooter tank. The desooter tank has the following main design components:
A provision of tar exit is needed to remove this tar slurry continuously out of the tank. Spargers are needed to form bubbles of exhaust gas in the mixture. Blowers are also attached to the system to compensate for the back pressure generated in the system at each step.
Now, we needed to condense the water vapor which flow along with exhaust gases out of this tank. This was required so that these excess water vapor do not clog the silica gel at the outlet of the tank. For this, we attach a heat exchanger at the outlet of the tank. It’s a cross flow type heat exchanger, with which we did some slight innovations to increase the efficiency of the condensation process. Cold water flows through the exchanger pipes and steam flows on the outside. We here filled the outside of the pipe portion of the exchanger with metal ball bearings. These ball bearings, through conduction, cool down to acquire the temperature(similar to) of the flow pipes and hence increase the surface area available to the vapor for condensation, speeding up the condensation process. The remaining exhaust, congaing NOx, Sox and some amount of water vapor now passes over the silica gel tank at the outlet of this desooter tank.
Studies showed that NOx and SOx, in the presence of water vapor, react over Alumina-silica gel(SiO2.Al203) to lead to an oxidation reduction reaction forming NO and H2SO4. This was a remarkable discovery as the NRFA bacteria we are working with, effectively reduce NO into harmless forms, and hence we needed NO as the output from this tank. Thus, the NO rich gas from this tank is passed into the next tank containing NRFA bacteria for further action. H2SO4 produced is extracted and used for industrial purposes.
Click here to view the 3D model
Tests were carried out on the prototype, taking in exhaust from a diesel burner and passing it through the system. The desooter tank worked even better than expected, collecting soot out from the exhaust (which was weighed after each run). NOx sensors placed at the edge of the silica gel tube confirmed that the gel was catalysing the reaction that we intended to do, converting NO2 to NO.
While working in the project, towards the end, the main complexity we faced was of Heme protein. It is so because most of the enzymes we were using were Cytochrome C Hemo proteins, which require a Heme group for thein 100% activity. While scavenging the research papers, we found out that nobody, till date had found a perfect solution for the over expression of Heme protein. Under the constraints of time, we managed to compile the literature, designed the perfect strategy and using Copasi (modelling tool), we found some novel pathways to regulate and manipulate the increase in Heme production and hence the activity of the Heme proteins. We did manage to extensively characterize the NrfA cloned gene, which served as the nitrate reductase. We found out that even without using any extracellular or intracellular Heme over production, our clones were highly efficient in reducing the nitrite and subsequently converting it into ammonia. Finally, we came to the conclusion that while expressing NrfA in a constitutive fashion, there was increased conversion of NOx to ammonia.
In the mechanical half of the project, we designed a prototype which in effect served as the perfect “Pollution Crusader”. We tested the model against the exhaust emitted by a diesel engine. Although the efficiency wasn’t that high, it did show positive results. We observed considerable reduction in the amount of NOx and SOx reduced. In a nutshell, our prototype model is capable of removing the harmful soot particles, gases like NOx and SOx and also the particulate matter present in the exhaust gases of the various engines being used. The prototype model designed will not only serve as an alternative for the existing technology, but has the potential to bring revolutionary changes in the upcoming days.