ECOS is a BioBrick designed to detect xylene. Xylene is a carcinogen in crude oil that is closely associated with two other aromatic hydrocarbons, benzene and toluene,that are the more dangerous compounds in crude oil. In Consort, Alberta, our economy is based almost entirely on agriculture and the oil industry. These two portions of the economy coexist side by side; oil wells are drilled on land adjacent to crops and cattle. This poses an issue if oil spills occur. While oil companies have strict regulations and protocols that they follow, it is always important that we increase our environmental stewardship. There is not a test currently that can check for contamination on site. There is little one can do after sending a sample to a lab and not receiving results back for weeks at a time. In order to properly monitor these sites, farmers and oil companies alike need the tools to do so. This is where we come it. ECOS will provide an on-site test that is cheap, efficient and easy. After talking to many community members we believe that our project could be used for semi-annual testing of sites, water monitoring and post spill monitoring.

ECOS consists of the following parts:

(Pic of sequence without reporter)

J23100 - which is a constituent promoter.
B0034 - the RBS for our XylR gene.
I723017 - the XylR coding region which encodes for the transcriptional regulator XylR protein.
B0015 - the double stop codon for this sequence.
I723020 - This is the Pu promoter.
B0030 - RBS.1 strong.

We also attached the Reporter AmilCP in the backbone pCB1C3 to give us an output in correspondence to the level of xylene present.

(pic of whole sequence)

Prototype 1:Animation

Alginate beads! We were hoping we were going to be able to trap our E.coli containing ECOS in the matrix of alginate beads. Alginate is easy to manipulate, it is cheap, light and safe. It would also be extraordinarily simple to operate as you simply place the beads in the soil or water and wait. We did several trials with alginate; experimenting with different protocols to form the beads. Unfortunately the beads ended up being totally opaque. With our trials with ECOS, we had to spin the cells down to see the AmilCP. You could not detect a colour change when the cells were suspended in the LB. With this information, there is no way we would be able to tell if the protein was being produced or not in the alginate beads. We also looked into making beads out of agar. Agar is too porous though and would not be able to hold the bacteria within the matrix.

(pic of both beads with labels)

Prototype 2: (Animation)

While this prototype is slightly more complicated, at this point we may have to sacrifice simplicity for something that will give us results. We designed this prototype so that it has a positive pressure source which creates a current for our xylene to travel from our sample, which is in a heated container so that the xylene is vaporized, to our ECOS container in which we bubble it through the E.coli culture to get results. This was a plausible idea, as it would require fairly non-expensive materials and could be maintained by the average business person.

Our Results

We were able to successfully create two parts. The first being just ECOS and the second having AmilCP attached. We did a lab on the part including AmilCP and did have positive results!

(Photo’s with labeling guide attached)

For more details please view our lab write up on the part!

(LAB WRITE UP goes here… it’s referred to in the notebook a lot)

Our Future Plans

As AmilCP is hard to see, we plan to look into finding a different reporter which will be easier to work with. Protein Paintbox has many different and vibrant proteins that may work better with ECOS. We have also discussed improving our prototype to make it as efficient as possible. A “seek and destroy” idea has also been brought up. This would mean we could not only detect the oil but also break down the harmful carcinogens contained in it.