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.


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

CsgA is an amyloid protein monomer that polymerizes to form long nanofibres. Assembled CsgA nanofibres are referred to as curli. CsgA accounts for the majority of the proteinaceous component of E. coli biofilms. CsgA is secreted from the cells, and in wild-type E. coli it self-assembles to form curli nanofibres tethered to the cell surface by the nucleator protein CsgB. CsgA can also polymerize without CsgB in vitro. CsgA is relatively tolerant of C-terminal peptide fusions, granted they are less than about 40 amino acids in length. As an amyloid, CsgA is rich in β-sheet structure and remarkably resistant to denaturing conditions.


Antifreeze proteins are a subset of ice-binding proteins. They adhere to the crystalline structure of ice by organizing ice-like water molecules on their surface. This makes further ice growth thermodynamically unfavourable. Although they vary greatly in structure, the ice-binding sites all have conserved properties. Here, we used the 37-residue, α-helical Type I AFP (we call it AFP8) from the winter flounder fish. We fused AFP8 directly to the C-terminus of CsgA. Most organisms that use AFPs circulate them in their blood as protection from freezing temperatures. However, AFPs can also be used to adhere to solid ice. In fact, the Arctic marine bacterium Marinomonas prymoriensis successfully uses an ice-binding domain as part of its large adhesin complex to keep itself at the top of the water column, where oxygen and nutrients are abundant [7].


As the name of this Gram-negative bacteria would suggest, it loves to degrade petroleum hydrocarbons. The species was first isolated from the waters of the Mediterranean, where it frequently forms oleolytic biofilms [3, 4]. The hydrophobic organic molecules that it degrades are used as a source of carbon and metabolic energy. We had originally considered directly fusing hydrocarbon-degrading enzymes (i.e. ethylbenzene dehydrogenase) onto CsgA. However, since individual enzymes function poorly outside of the cell, we opted to append whole M. hydrocarbonoclasticus bacterial cells instead. When it comes to optimal hydrocarbon degradation, you can’t beat Nature!


SpyTag and SpyCatcher form two halves of a split protein system. When these two domains come together, they spontaneously form a new covalent peptide bond. This permanently links SpyTag and SpyCatcher, along with whatever was attached to them. We used this system to overcome CsgA export size limitations. Nguyen et al. found that CsgA with fusion peptides larger than approximately 40 amino acids were too large to be properly exported [5]. However, SpyTag is only 13 amino acids long, so we used CsgA-SpyTag fusions to enable us to add larger domains than could otherwise be exported. Our Lectin-SpyCatcher fusion would then be added to the exported CsgA-SpyTag, permanently appending our large (30 kDa) Lectin to CsgA.

Sugar Binding Domains

Sugar binding domains are used to tether the hydrocarbon–degrading M. hydrocarbonoclasticus (MH) to the CsgA scaffold via dextran. Not enough is known about MH physiology to make genetic modification (i.e. expression of SpyCatcher) of this exotic organism practical. Thus, we made use of an existing MH surface protein: a sugar binding domain. The sugar binding domain is a part of Region III of the large MhLap adhesin complex expressed on the surface of MH. Our affinity chromatography and microfluidics experiments proved this domain binds dextran. A lectin domain was appended to CsgA to enable the curli biofilm to latch onto dextran as well. Dextran thus serves to crosslink MH to the E. coli CsgA biofilm. We chose the C-type lectin from residues 403-532 of the human polycystin-1 protein, encoded for by the PKD1 gene [6]. Lectins bind sugar moieties of many polysaccharides, playing key roles in cellular recognition in many organisms.