Team:Queens Canada/Overview

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THIS IS THE PROJECT OVERVIEW PAGE

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.

Marinobacter hydrocarbonoclasticus

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/SpyCatcher

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, permanently appending our large (30 kDa) Lectin to CsgA.