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Team:Consort Alberta/project
From 2013hs.igem.org
Our Project:
Our project has been the development of ECOS (Environmental COntaminant Sensor). The heart of the sensor is an E. coli culture that has been modified to produce green fluorescent protein when exposed to xylene. Xylene was chosen as a trigger because its presence is very well correlated with the presence of other more dangerous compounds such as benzene and benzene derivatives. This class of compounds is able to intercalate into DNA, causing mutations and is carcinogenic as a result. The XylR transcriptional activator is a protein which in the presence of m-xylene will bind to the Pu promoter resulting in the expression of our reporter, green fluorescent protein. The other components of the sensor include a heated sample container to assist in vapourizing the xylene and an air pump to push the vapour through our bacterial culture.
Application/Technology:
Components for the application of our project:
1) Power source – to power the heater and the pump.
2) Heater – to heat up our sample so that it vaporizes the xylene in the sample so that it can be transferred to our E.coli more efficiently.
3) Pump – to create a slight vacuum or pressure so that the aromatic xylene is bubbled into our “E. coli box.”
4) E. coli box (sealed) – to hold the E. coli as well as keep it in a dark environment so that we can see the fluorescence. It would have a sealed hole on the side for the UV flashlight as well as one on the top to see the fluorescence (also a sealed hole).
5) UV flashlight – to make the GFP fluoresce.
6) Tubing – to connect the heater to the “E. coli box” and the “E. coli box” to the pump.
7) Funnel – this will act as a lid for our heater/sample, tubing will be connected from the funnel to the “E. coli box.”
8) A wavelength detector – to differentiate the UV and the green fluorescence wavelengths to make sure that there is GFP in the solution (GFP indicates the presence of xylene)
The most practical way of doing this would be to have a vehicle's DC power outlet as the power source, so that you can take the components out to the oil well in question and do your test right then and there. This is more efficient than sending it to a lab to have it tested because xylene only has a half life of about 18 hours. This means that the longer you take to test it, the more of the xylene that will degrade, which in turn will give you false or inaccurate results. Also you will need a heating device that is compatible with the power outlet on your vehicle. A travel coffee mug will work nicely for this because it is light, cheap, portable, and a funnel can easily be placed on the top of it. Afterwards you would attach the funnel or lid to a tube which leads to the “E. coli box”, this tube will be submersed deeply into our E. coli solution. This is so that the aromatic xylene will be exposed more effectively to our E. coli, thus giving better results. To cause this bubbling you will need to create a vacuum, which can be done by using a pump (which can also be powered by your vehicle). The intake of the pump would be attached to the “E. coli box” so that it is not sucking up any of the E. coli solution. The “E. coli box” itself will need to be encased in a black box so that the fluorescence will be visible. This box will have an eye hole and, on one side, a UV flashlight will be attached so that the GFP will fluoresce. Since the green fluorescence might be hard to see with your eyes, we would have a wavelength detector so that you can differentiate the UV and the green fluorescence.
How this relates to us:
Each and every day on the way to town or even to my neighbour’s house I can see several oil wells. This concerns me because many people in agriculture have to work around these oil wells. Xylene, benzene and toluene all vapourize easily and, even though they do have a strong smell, most farmers still have to get their job done and it only takes a little exposure to these compounds (1 ppm/8 hour day is permissible) each day to accumulate enough to become hazardous. As well, plants and cows don’t mind the smell too much. At the point at which it becomes hazardous it is usually too late to do much because liver problems, birth defects and increased risk of cancer have already set in. We are making an E. coli that can detect this hazard before it becomes a problem to people, and hopefully we can send people in to clean it up. It also relates to the world because these gases get into plants, then into the cows we feed it to, which get shipped worldwide, as food for human consumption.
E. coli Plasmid Design:
Here is how we are going to detect xylene with our plasmid sequences.
Components for our first plasmid DNA sequence (Total size is 5708Bp):
1) BBa_I723021 (2282Bp) (created by iGEM07_Glasgow) – which is a composite part containing the Pr promoter, BBa_I723018, which is the naturally-occurring (possibly stationary-phase only) promoter for the XylR gene (which encodes the transcriptional regulator XylR). The next part in this DNA sequence is the RBS for XylR, BBa_I723019, also the naturally-occurring RBS found in the regulatory region of the XylR gene. Next is the actual XylR coding region, BBa_I723017, where the protein XylR is synthesized. The protein encoded by XylR recognizes the chemical xylene (a petroleum derivative commonly found as a pollutant near industrial sites) and undergoes a conformational change allowing it to bind to, and positively regulate, the Pu promoter. XylR, therefore, should be used in combination with Pu promoter to achieve a switch that activates in response to xylene. Lastly this sequence has the double terminator, BBa_B0015, which is used as the stop codon for this sequence.
2) pSB1AK8 (3426Bp) – this is the plasmid backbone we will be using for our plasmid. It has both AmpR and KanR which are the bacterial resistance genes to ampicillin and kanamycin. We need bacterial resistances so that we can use those antibiotics to kill off any unwanted E. coli or other invading bacterial without harming our desired E. coli. Along with these resistances, there is a kill switch attached to the backbone. This kill switch, BBa_P1010, has a half life of 2 hours, which means that in 2 hours about half of the cells should be dead.
This plasmid is responsible for producing the transcriptional regulator XylR which will bind to xylene and promote our next plasmid.
Components for our second plasmid DNA sequence (Total size is 4610Bp):
1) BBa_I723020 (320Bp) – this is our Pu promoter, when the protein encoded by XylR comes in contact with xylene it goes through a change allowing it to bind to, and positively regulate, the Pu promoter. Basically it starts the process of our RBS to initiate the transcription of our DNA sequence to mRNA and then through translation the GFP is synthesized.
2) BBa_B0030 (15Bp) – our next piece, RBS.1 strong, is where the actual transcription will start.
3) BBa_E0040 (720Bp) – the GFP wild-type, this is the gene that causes our E. coli to glow when exposed to UV light.
4) BBa_B0015 (129BP) – lastly we have our double terminator, which acts as our stop codon for this DNA sequence.
5) pSB1AK8 (3426BP) – same backbone as before so that we don’t accidentally kill off any of our wanted E. coli
This plasmid is our reporter for the presence of xylene which in turn tells us other hydrocarbons are present.
Our first plasmid is essential to the way our xylene detector is going to work, because the XylR sequence will constantly be producing transcriptional regulator XylR. When the transcriptional regulator XylR is in the presence of m-xylene, a benzene with two methyl substituents, it undergoes a conformational change which allows it to bind to m-xylene, which then allows it to positively regulate the Pu promoter starting the production of GFP, which is our reporter gene for xylene.
The reason why this is useful is because more harmful aromatic hydrocarbons are going to be found with xylene but will be more prominent than xylene will be. Examples: benzene and toluene, meaning that if there is xylene in an oil spill then there will be more toluene and even more benzene. Now xylene isn’t very toxic considering that its half life is only 18hrs and its LD50 is about 4000mg/kg. Even though toluene only has a half life of about 13hrs, the reason why it’s so much more dangerous is because if you’re exposed to it, it’ll make you feel light headed and if exposure is continued you’ll pass out, and eventually you’ll die. These symptoms will continue until it leaves your system -- the problem is it can be stored in your body’s fat for 1-3 days. The LD50 for toluene is about 700mg/kg. Benzene is the worst of the three, since its half life can range from 1-21 days as well as its LD50 can be as little as 930mg/kg. Some of benzene’s symptoms include birth defects, increased risk of cancer, bone marrow failure and liver defects. A quote from American Petroleum Institute (API) - “it is generally considered that the only absolutely safe concentration for benzene is zero.”
