Team:Queens Canada/Overview


The Arctic is predicted to contain approximately 22% of the world’s oil and natural gas resources locked up beneath its basins. Due to the lack of natural resources in the surrounding tundra biome, bioremediation of oil spills can be especially challenging in the Arctic. As a result, an effective and environmentally safe method of petroleum cleanup may come in high demand during a major oil spill, which has been shown to incur costs ranging from the hundred millions to billions of dollars for conventional containment and cleanup.

The QGEM Team is turning to nature as our inspiration for building a safer and cheaper method of oil spill cleanup, using synthetic biology as our tool. This summer, we will be designing and engineering a bacterial biofilm-based material that functions to bind ice and recruit oil-degrading native marine bacteria. This will be done by engineering a protein naturally expressed in bacterial biofilm, CsgA, to append an ice-binding protein and a bacterial adhesin domain. The end product will be a dynamic, bifunctional biomaterial that may be deployed into the Arctic marine environment during oil spills as a bioremediation factory, with limited disturbance to the surrounding ecosystem


Agar plate streaked with negative control device (left) and positive control
device (right) viewed under UV light. Looks like they work!





The Arctic is predicted to contain approximately 22% of the world’s oil and natural gas resources locked up beneath its basins. Due to the lack of natural resources in the surrounding tundra biome, bioremediation of oil spills can be especially challenging in the Arctic. As a result, an effective and environmentally safe method of petroleum cleanup may come in high demand during a major oil spill, which has been shown to incur costs ranging from the hundred millions to billions of dollars for conventional containment and cleanup. The QGEM Team is turning to nature as our inspiration for building a safer and cheaper method of oil spill cleanup, using synthetic biology as our tool. This summer, we will be designing and engineering a bacterial biofilm-based material that functions to bind ice and recruit oil-degrading native marine bacteria. This will be done by engineering a protein naturally expressed in bacterial biofilm, CsgA, to append an ice-binding protein and a bacterial adhesin domain. The end product will be a dynamic, bifunctional biomaterial that may be deployed into the Arctic marine environment during oil spills as a bioremediation factory, with limited disturbance to the surrounding ecosystem










Biofilms


In nature, the majority of bacteria exist as biofilms. Biofilms are organized bacterial communities that grow on an extracellular matrix scaffold composed mostly of polysaccharides, proteins and nucleic acids [2]. Biofilms typically carry negative connotations, particularly in medical settings where they are associated with antibiotic-resistant infections. However, we can exploit the same traits that make biofilms a formidable healthcare challenge to create engineered biomaterials. Bacteria in biofilms have several characteristic advantages over their planktonic (free-living) counterparts [2]:

  • Enhanced resource capture and processing
  • Facilitated social communication (quorum sensing)
  • Protection against environmental stress (i.e. antibiotics, shear stress)


Our engineered E. coli (expressing our CsgA fusions) can provide the oil-degrading M. hydrocarbonoclasticus with an ice-binding biofilm scaffold, significantly increasing its ability to survive and degrade hydrocarbons in harsh Arctic conditions.



CsgA Operon



absorbance at 600 nm
Fig 1. Fluorescein standard curve.

The above fluorescence calibration curve (Fig. 1) was created by measuring fluorescence intensity of different concentrations of fluorescein.

Fluorescents of the test devices.
Fig 2. This graph shows the fluorescence of each test device measured every two hours over a span of 6 hours.

Cultures were sampled at the += 0,2,4,6 hour marks in 500ml aliquots from 10ml cultures. All samples were added to a 96-well plate to measure fluorescence intensity. Fluorescent values were normalized prior to plotting. All points on the graphs are the average of the two colonies grown (which themselves are the average of 4 wells each). Test device 2 had the greatest overall increase in fluorescence. Both the negative control device and the LB+ chloramphenicol sample had no significant increase in fluorescence.


absorbance at 600 nm
Fig 3. This graph shows the absorbance at 600 nanometres of each cell culture, which
provides an estimate for the number of cells in the samples.

Every test device exhibits steady, somewhat sigmoidal bacterial growth. The LB + chloramphenicol sample shows no change in OD600 over time.

Conclusions


  • The Queen's_Canada iGEM team was grateful for the opportunity to contribute to the Interlab Study for the first time.
  • It appears that our cells only began expressing significant amounts of GFP after the 4-hour mark. One would expect the curve of increasing GFP fluorescence to mirror the curve of OD600, if GFP expression is truly constitutive. The OD600 curve shows steady, somewhat sigmoidal growth, while the fluorescent intensity curve is a plateau until after 4 hours have elapsed.
  • This suggests either a certain threshold concentration of GFP is required to be detectable by our plate reader, or that GFP expression only begins at a certain cell density threshold (which is reached at approximately 0.2 OD on our Figure 3 graph).
  • Both the LB + chloramphenicol and negative control wells showed no significant increase in fluorescence, as expected.

References


  1. Kwok, R. 2010. Five hard truths for synthetic biology. Nature, 463, 288.
  2. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. 1994. Green Fluorescent Protein as a Marker for Gene Expression. Science: 263(5148), 802-805.