Team:Consort Alberta/Parts
ECOS
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 found in crude oil. In Consort and most of 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 risk to consumer health and to our economy 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. 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 that are intimately involved in agriculture and the oilfield we believe that our project could be used for semi-annual testing of sites, water monitoring and post spill monitoring.
Our BioBrick:
ECOS consists of the following parts:
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.
Design
Prototype 1:
Our first prototype involves trapping 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. We can manipulate the density of alginate's matrix to allow us to entrap our bacteria in the beads. The proteins will still be able to get through the pores so we can see an output. 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 color 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. However, the alginate's matrix would still allow the xylene to get in, which ensures our beads are safe. 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.
Prototype 2:
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.
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!
For more details please view our Lab Write Up
We created a rudimentary prototype of this three-chambered system to test our E. coli and xylene soil samples with. The prototype consists of a 2L milk jug that is divided to contain our soil sample, a double fan system that keeps the air circulating between the milk jug and the mug with our E. coli in it. As outlined in our Notebook, in order to prepare for our "real life" experiments, we mixed 3.5mL of xylene for 350mL of soil. We then added the soil to our built prototype and added LB and cell cultures from yesterday to the chamber that holds ECOS. We then let it sit overnight, starting at 4 p.m. on the shaker table until 10 a.m. the next morning (a total of 13 hours).