Team:METU-Ankara

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Introduction

MethanE.COLIc project is designed to solve one of the problems of Turkey on worker security in mines, by constructing the natural parts of organisms.The main reason for us to choose such a project is that in Turkey- and also in many countries- each year, huge numbers of workers in mines lost their life due to deficiencies and conditions in their working areas (mines). The release of Grizu gas leads to subsidence of mines and so death of workers.This project is also designed to solve one of universal problem; global warming. Methane gas is a potential greenhouse gas.This project focuses on sensing the ambient methane gas and converting to one of the bio-fuel sources, methanol. Methanol is entrapped by product of one of the constructs of project, and then cells are dead by kill switch device of project to elute methanosol.

Steps

• Methane sensing
• Conversion
• Entrapment
• Kill switch

Team

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WET LAB

Whole summer in lab work we aimed to design 7 new E.coli compatible Biobricks and 1 composite part from 2011 kit plate distributions, also we aimed to characterize each part by protein analysis and fluorescence protein measurements.

We achieved design and compatibility of parts in E.coli, however not all parts’ characterization is achieved. Only the genes of designed composite part from kit plate is characterized by GFP measurements.

In this section you can find out more details on all our wet lab works. You can also find our data under results subtitle and information about design of our parts-biobricks with explanations in literature. It also contains materials and protocols, and finally There is safety questions list on our safety considerations both in the lab and on a wider environmental scale.

Overview

MethanE.COLIc project arises from sustainty and conservancy of human health and safety. Therefore while we were constructing the organismal device we also wanted to inform people from all age and we wanted to increase the public awareness on our project; methane gas based (firedamp) explosions and the greenhouse effect of methane gas.

Our aim is to inform people in each age. After brainstorming and searching, we planned the content of information and activities according to following subtitles.

Future Work

Methane explosion is the main problem since the very beginning of the coal mines. All countries tried different systems to solve this methane problem. When we decided to join iGEM 2011 competition firstly, it was very hard to chose a project from our brainstorms. All member of this group come up with a very reasonable idea for our team and we evaluate them all were seemed very beneficial for humanity. However, one day we face the problem of methane explosions on the news and at that time we decided to create new idea for this problem. After a long literature research, we found how seriously dangerous the methane gas and how people deal with this big problem on coal mines

1. Australia
2. Belgium
3. Canada
4. Chile
5. China
6. France
7. New Zealand
8. Poland
9. Russia
10. Australia
11. United States

These are the countries which have been faced with too big methane explosions true out their history. After a certain research we found that to deal up this problem, companies spent lots of money for effective solutions.
Regenerative Thermal Oxidizer from MEGTEC capable of handling 60,000 scfm of VAM. At a methane concentration of about 0.7% a unit this size generates 50,000 carbon credits per year. This unit costs about US$ 1,5 million Elizabeth Obediente from CO2IMPACT at a methane upgrading facility at a coal mine in Alabama. The gas is stripped of impurities and upgraded to 96% methane per volume and then compressed and then sold to the natural gas pipeline. Ventilation Air Methane (VAM): VAM units cost about $20-$30 USD per scfm.

At some mines, downtime is considerable, causing expensive losses to the mine. Ventilation cost cuts can be up to half of the original cost of the ventilation system. In one study, a mine with 400 ft3/ton of methane using vertical wells to pre-drain will result in an estimated US$ 11 million over 20 years in reduced ventilation costs, not including carbon and energy revenue[iii] (U.S. Environmental Protection Agency, 1999). Like many solution system for methane problem, the tree examples of solution of methane problem did not satisfied us, so we created our simple and very effective way to deal with the methane.( http://www.co2impact.com/cmm.html )

According to our methane E colic project, we aimed:

• Save the coal workers’ life
• Save the nature
• Process a new energy
• Cheap system

Save the coal workers’ life

When we produce our project, we stop the search the worlds system and turn back to our country, so we saw that methane (CH3) is a big problem to get rid of our mines too. The way carry out on our county has some missing parts on safety. While we were proceeding our project, we focus on this part mainly and we have shaped it according to this.
We aimed to collect the methane gas from coal mines to the tanks which are filled with water. When the methane passes the legal percent of %1, according to our project, we dissolve the gas on water tanks. After that we put tablets which include our bacteria on the tanks , so the methane and methanol process will begin with this step.

Years	Death workers on Turkey
1983	103
1990	330
1995	40
2003	17
2004	3
2005	18
2006	21
2007	7
2008	43

According to Prof. Dr. Mustafa ÖZTÜRK’s research (Vice President of Parliamentary Commission for the environment on turkey), this the death workers amount since 1983 to 2008 on Turkey’s coal mines.
Our way, collecting all methane gas from the mine to tanks, not only make methane free working space for workers, but also could make the space more workable and safer.

Save the nature

During our research for safety systems for methane all over the world, we saw that in most ways, methane release to the atmosphere with or without some elements and chemicals. Just a few systems are used methane to produce new energy or something else.

We noticed that methane gas is twenty four times harmful than the carbon monoxide gas for the atmosphere. According to our project, on the way that converting methane gas to methanol form, we prevent methane to make any damage to the nature on gas form.

Process a new energy

During the research process for our project, we also realized that energy is the most important thing on today and future life. Today, all energy sources are almost over including petroleum. People are searching new energy source. As the last news from china, everyone saw that nuclear energy is not a secure and trustable way to get energy.

On this century, people understand the nature’s value for human life. Now renewable energy systems are built all around the world like wind turbines and sun batteries.

According to our project, we planed that methanol could be used for making bio-fuel instead of ethanol. For the bio-fuel, ethanol is collecting from especially from the corps. However, on this way, we use corps unnecessarily. Studies suggest that vehicles that run on 85 per cent methanol blends, as well as petrol, actually have performance capabilities that equal or surpass vehicles running on petrol alone.

Also, according to Dr. Robert Zubrin, Methanol is cheaper than ethanol. It can also be made from a broader variety of biomass material, as well as from coal and natural gas. And methanol is the safest motor fuel.(American enterprise, February 13th, 2006)

Cheap system

There are lots of system use now for methane gas and the gas explosions on mines. However, when we look closer to them, they are too expensive to build for a mine. If we notice also there are global economic problems nowadays, it seems not possible that companies may not be to get enough precaution for their workers, even their own life especially on the developing countries.

On the other hand, we put all the bacteria that we produce in a capsule tablet. They are totally harmless on this form. When we put them in the water tanks, they will come to life and start their work.

This process does not cost million dollars or does not get a lot of time for building very big facilities. It is very cheap and has very easy process time.

Our project is open for research and development. For the future of it, we planned to put smelling part to our bacteria for making workers area more fresh and comfortable. In addition, we can design a system for our bacteria to send it in the mine and maybe start the process inside the mine. Furthermore, we put also biosensor part the bacteria and the bacteria measure the percent of the gas level with itself.

PartnerShip

Videos

In Africa 1999

• July 30 1999

In Ukraine, 2001

• 20 August 2001

In UK, 2006

• 20 June 2006

In Siberia, 2007

• 19 March 2007,

In Turkey, 2009

• 11 December 2009

In Turkey, 2010

• 20 February 2010
• 18 May 2010

In Chile, 2010

• 5 August 2010

In Russia, 2010

• 9 May 2010

In New Zeland, 2010

• 22 November 2010

In Virginia, 2011

• 19 May 2011

In Ukraine, 2011

• 29 July 2011

In Mexico, 2011

• 4 May 2011

In Pakistan, 2011

• 22 March 2011

Team

Methane sensing

i) Introduction

In our methane.colic project, the first critical point is to sense the ambient methane gas. While in literature search, we have focused on the methane and DNA or methane and regulator protein interaction which binds to DNA after binding to methane. In structure, methane consists of one carbon atom and four hydrogen atom which means it is smaller in size and weight. Therefore it is hard to find any study based on methane interaction with DNA or protein to regulate transcription. This made us search for carbon hydrogen bond interaction with any oligo or protein to trigger transcription of any metabolite product. So that we also searched the literature for any organism which utilizes any alkane chain and alkane which regulates the transcription of organismal metabolites by interaction with DNA or protein. Since bacteria are environmental adapting organisms, it is possible to find any alkane to regulate the transcription of degradation metabolism. Then we have found the organism, Pseudomonas oleovorans to analyze the alkane degradation of organism for its carbon source.

ii) Background

Many microorganisms live in the environments where the conditions changing frequently and so the evolution is inevitable for mechanisms to withstand unfavorable situations. Therefore microorganisms can use their specific and sensitive mechanisms for sensing the required nutrients for them or any pollution to affect their sustainity. Otherwise they can expose to mutations which change the gene expression and gain new functionalities. In case they have ability to survive in such conditions.

While we were scanning the literature based on methane and alkane degrading organisms we have found some organisms that sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.

We analyzed the soil bacteria, Pseudomonas oleovorans. This strain can assimilate the alkane for its carbon source and one of the microbial whole cell –biosensor. They have a gene cluster which codes for degradative pathway and includes the activator which interacts especially with linear alkanes. This activator protein, AlkS, in the presence of alkanes, induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes. We have analyzed the related articles in which the promoter region is studied and showed that it is expressed in E.coli correctly because of the corresponding RNA polymerase binding regions.

REFERENCE:

1. Canosa, I. & Yuste, L. (2000). A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. Molecular Microbiology, 35 (4), 791-799.

iii) Modelling

Since the parts that we have found belongs to yeast organism, it was hard to us to manipulate it in bacteria E.coli. We designed the tests of this part by characterizing one of the subunits of methane monooxgenase (MMo). For the functional activity of monooxygenase enzyme, there are required 3 subunits as A,B and C. The A subunit of methane monooxygenase is 210 kDa protein that is supposed as the methane binding active site of monoxygenase complex. This protein consists of non-haem iron and contains p-hydroxo bridge structure. We planned the experiments of this step as characterization of protein A by PCR amplification with specific primers of full construct device to obtained the sequence of protein A. The methane gas is applied to cell culture with our modelled device then the cells were centrifuged and the gas concentration is measured from supernatant.

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i) Introduction

IWhen examined in order, conversion is the second step of device system. While in literature searches, it is found that methanotrophs which live in either extreme conditions or in deep oceans, the process is the same, methane is converted to methanol which is the oxygenated form of hydrocarbon group. The searches directed us to organism Methylococcus capsulatus which is one of the mostly studied organisms among methanotrophs. We designed the conversion part of this organism and modified coding operon according to E.coli strain to enhance the expression. The monoxygenase coding part with protein A, B and C products are designed for conversion step of this project.

ii) Background

Methanotrophs - methane oxidizing bacteria- are ubiquitous in the environment and play an important role in mitigating global warming because they are a unique group of gram-negative bacteria that grow aerobically on methane as sole source of carbon and energy (Hanson and Hanson 1996). They are also potentially interesting for industrial applications such as production of bulk chemicals or bioremediation. Methanotrophs converts methane to methanol and subsequently to formaldehyde which is assimilated or further oxidized to CO2 for biosynthesis of cellular forms.In this biosynthesis pathway the conversion of methane to methanol is catalysed by methane monooxygenase (MMO) which is a non-specific enzyme complex that also catalyse the oxidation of a wide range of aliphatic, alicyclic and aromatic compounds (Colby et a l . , 1977; Higgins et a l . ,1979). The first step in the oxidation of methane to precursors of organic compounds is the conversion to methanol by methane monooxygenase, the key enzyme, which exists in two forms: the cytoplasmic, soluble methane monooxygenase (sMMO) and the membrane-bound, particulate methane monooxygenase (pMMO). sMMO components have been expressed in heterologous and homologous hosts. Therefore we searched more on soluble form of monooxygenase enzyme. When analyzed in detail, pMMo differs from sMMo by copper ion concentration. Copper ions have been shown to play a key role in regulating the expression of both MMO enzyme complexes. When its concentration increases the differentiation goes through particulate form. Here we analyzed the soluble form of methane monooxygenase to express in heterologous host.

a) Soluble Methane Monooxygenase

In contrast to pMMO, sMMO has extremely broad substrate specificity and can oxidise a wide range of non-growth substrates such as alkanes, alkenes and aromatic compounds thus making it the more attractive enzyme for co-oxidation reactions. sMMO is expressed only under conditions in which the copper-to-biomass ratio is low, i.e. under “low-copper” growth conditions, when copper ions are omitted from the trace elements solution of a standard mineral salts medium or cells are grown in a fermentor to high cell densities.There is also some evidence that copper ions inhibit the activity of sMMO (Jahng and Wood 1996). Like many other multi-component oxygenase systems, sMMO contains a component of approximately 16 kDa, Protein B, which serves an “effector” or regulatory role. The activity of Protein B may be regulated by proteolysis at its amino terminus (Lloyd et al. 1997). At low concentrations, Protein B converts the hydroxylase from an oxidase and stabilizes intermediates necessary for oxygen activation. Saturating amounts of Protein B dramatically increase the rates of formation of intermediates and accelerate catalysis of methane to methanol by sMMO (Lee and Lipscomb 1999). When analyzed, the most extensively characterised sMMO enzymes are those from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b (reviewed in Lipscomb 1994; Deeth and Dalton 1998). Therefore we preferred to study on Methylococcus capsulatus (Bath) for monooxygenase.The sMMO is a non-haem iron-containing enzyme complex consisting of three components. The hydroxylase consists of three subunits of 60, 45 and 20 kDa arranged in an α2 β2 γ2 configuration. sMMO genes are clustered on the chromosome of Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. mmoX, mmoY and mmoZ encode the α-, β- and γ-subunits respectively of the hydroxylase. mmoB and mmoC code for Protein B and the reductase component. Interestingly, mmoB lies between mmoY and mmoZ; an ORF of unknown function, designated orfY, with a coding capacity of 12 kDa, lies between mmoZ and mmoC in all genes clusters analysed to date (McDonald et al. 1997).

REFERENCE:

1. J. Colin Murrell · Bettina Gilbert · Ian R. McDonald, (2000), Molecular biology and regulation of methane monooxygenase Arch Microbiology(2000) 173 :325–33
2. Colby, J . & Dalton, H. (1978). Resolution of the methane monooxygenase of Methylococcus capsulatus (Bath) into three components. Biochemical Journal 171,461-468
3. Hanson Te&Hanson RS(1996) Methanotrophic bacteria.American Society forMicrobiology.Microbiol. Rev., Jun 1996, 439-471, Vol 60, No. 2
4. Charlotte, A., West, G. & Horward, D. (1992). Functional expression in Escherichia coli of proteins B and C from soluble methane monooxygenase of Methylococcus capsulatus (Bath. Journal of General Microbiology, (138), 1301-1307.

iii) Modelling

For this step of MethanE.COLIc project, the experiments were planned and modelled as protein characterizations. The protein B and C were previously characterized and analyzed proteins in literature. Based on this publications we planned the experiments as purifying protein from cell extract then SDS-PAGE analysis.

Workshops

5-10 year old group

For this age group, we prepared and modified one of the national child game. In this game there is a circle of group of child, one is not included in that circle. It runs around the circle and labels one of the participants in circle. When recognizes, it chases that one with singing that special song. In our modified game, the one that out of circle was our restriction enzyme due to our group in video, we assigned the enzymes into two,also they are double digestion enzymes. These ones wore white caps in video. And we have three insert groups which wore red, green and yellow caps. The blue capped ones were our backbone.
During (By this game and at the begining of) this game we told to kids what is DNA, enzyme and insert, and what we have done as Synthetic Biology.

10-17 year old group

This group consists of high school aged children groups.Through out the workshop, we have paired them as two groups. Firstly we have explained Synthetic Biolgy and what Synthetic Biology is able to do. After these training, we told them our project and taught them basic molecular biology procedures and what are required for basic synthetic biology based experiments. We have done the experiments(cloning procedures) together and also they did individually. We also encouraged them to join iGEM High School division.

18-24 year old group

This group consists of METU students. We arranged the Physics lectures in METU by contacting with instructor. To give an effective presentation (Due to physics is an service course in METU), we have reached to students in several departments. We had told our project individually and told them what is Synthetic Biology, we gained many volunteers to join METU-iGEM team for the following years. Also in METU we have an online lecture application as METU open course ware. For this web page, we have prepare the course notes and they were uploaded to reach many students from all around to inform and contact for further relations about Synthetic Biology.
Moreover, we distributed hand outs about our project and synthetic biology at METU campus, when the new students registrations were done. We aimed to inform students about our project and synthetic biology to attract their attention on the issue.

Online lecture link:&nbsp: http://ocw.metu.edu.tr/course/view.php?id=137

Above 24 year old

For this group of people we met with group of engineers in Turkey who work in mining fields in Turkey. We have told our project in details and we have consulted them on background of our project. What are the conditions in mines, what are the general regulations for worker safety and what are the rooted controls for any gas release are the points which we asked and discussed on.

List of Engineers:

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Entrapment

i) Introduction

After conversion of methane to methanol, the successive step is the entrapment of methanol. Methanol is an alcohol form of hydrocarbon methane and includes hydroxyl group. Hydroxyl radical groups are highly reactive and, and so short-lived; however, they form an important part of radical chemistry. Hydroxyl groups are especially important in biological systems and their chemistry because free radicals tend to form hydrogen bonds both as donor and acceptor. This property is also related to their ability to increase hydrophilicity and water solubility. Therefore hydroxyl free radicals cause damage to oxidative cells and cellular membranes. In order to produce the methanol for further industrial manufacturing in cellular organism, the organismal conditions should be adjusted. In the light of this, for our bacterial system we transferred the protein coding sequence(named as LUSH) of Drosophilia melanogaster to attack to free methanols and hydroxyl group. Here is some structural information about protein LUSH.

ii) Background

LUSH is an alcohol-sensitive odorant binding protein expressed in the olfactory organs of Drosophila melanogaster, and it is used as a model system to investigate the biophysical nature of alcohol-protein interactions at alcohol concentrations that produce intoxication in humans. In this study, by using NMR spectroscopy, they have identified the regions of LUSH that show increased conformational stability on binding alcohols. These residues primarily line the alcohol-binding pocket. A direct measure of the degree of stability that alcohol imparts on LUSH has been provided. LUSH was originally identified as responsible for mediating an avoidance response to short-chain n-alcohols.

The general structure of odorant binding proteins consists of six α-helices surrounding a hydrophobic ligand- binding pocket which differs in size and shape between each protein. All these odorant binding proteins have a set of six cysteines that form three conserved disulfide bonds. In the study, by observing the X-ray crystal structures of LUSH-alcohol complexes, it was found that alcohol binds to a single site in the protein formed by a network of concerted hydrogen-binding residues located at one end of hyrophobic pocket. This binding site has some sequence and/or structural similarities to regions of several ligand gated ion channels (LGICs) that have previously been implicated in inferring sensitivity to alcohol. It is hypothesized in the study that the alcohol-binding site in LUSH may represent a more general structural motif for functionally relevant alcohol-binding sites in proteins. The characterization of the effects of n-alcohols on the structure and stability of LUSH is presented. Also, in the absence of ligand, LUSH exists in vitro in a partially unstructured state and binding of alcohols shifts the solution conformation to a more compact folded state which is accompanied by an increase in the overall protein stability. Those regions of the protein that show the largest changes in local dynamics on binding alcohol have been identified and it have been shown that these are predominantly associated with the residues that line the alcohol-binding pocket. The results provide a quantitative measure of the ability of short-chain alcohols to stabilize protein structure at physiological relevant concentrations.

REFERENCE:

1. Bucci, B. K., Kruse, S. W. & Thode, A. B. (2006). Effect of n-Alcohols on the Structure and Stability of the Drosophila Odorant Binding Protein LUSH. Biochemistry, 45 1693-1701
2. Thode, A. B., Kruse, S. W. & Nix, J. C. (2008). The role of multiple hydrogen bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH. J.Mol Biol, 376 (5), 1360-1376

iii) Modelling

In MethanE.COLIc project, LUSH protein is designed to hold methanol; the conversion product of methane. Methanol is bounded to LUSH protein through its hydroxyl group. In device experiments of this part, we planned to isolate and denaturate protein and then refold in presence of methanol and we planned to analyze the alcohol bounded protein in X-ray diffraction crystallogrophy.

Notebook

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Kill switch

i) Introduction

In this project the last step is the death of our bacteria to elute the protein(LUSH) bounded product methanol which is one of the precursors of biofuel. In our modified bacteria, the death of cells occurs by temperature dependent cell lysis device. The expression of this device takes place at 42 C since there is an RNA thermometer part in upstream of this device. At the downstream of this device there is an lysis casette. When transcription initiated at 42C holin and endolysin is expressed from this device and make cell dead.

ii) Background

This composite part consists of promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter (BBa_I712074) which is strong promoter from T7 bacteriophage. For strong transcription T7 promoter expression system is chosen. In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. RNA thermometer (BBa_K115001) is temperature sensitive that at 42C translation initiates. That means, up to 42 C RNA thermometer is in dimer form and at 42C it becomes linear. In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. The last ligate to this composite is bacteriophage 21 lysis casette (BBa_K124003). This lysis casette induces lysis in E.coli. This gene cassette consists of the S protein (Holin), R protein (Endolysin), and Rz protein. When translation initiates at 42 C then the following coding gene begins to produce the proteins which lyse the cell.

iii) Modelling

In MethanE.COLIc project, this composite is modelled to lyse and kill the bacteria when switched on at 42C. In experimental design, we ligated this composite without lysis casette (with promoter, ROSE and GFP) We planned two control groups. One is T7 promoter and GFP ligate, this is designed to measure expression level of T7 promoter by reading the GFP expression. The second control group is T7 promoter, ROSE and GFP ligate, this is designed to measure the expression level of T7 promoter with ROSE ligate at 37 C. The device experiment is done with T7 promoter ROSE and GFP ligate at 42 C in which the GFP expression is expected.

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