Team:Queens Canada/Description
Project Overview
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]. They have the capability of forming on living surfaces (e.g. dental plaque, plants) or nonliving surfaces (e.g. rocks, hospital settings). A recent endeavor being pursued by research labs around the world is the modification of key scaffolding components in biofilms to generate biosynthetic, artificially-functioning biomaterials. In E. coli, curli amyloid nanofibres are the primary proteinaceous components of the biofilm. Curli is formed by the self-assembly of individual 13-kDa CsgA protein units outside the cell [1]. Since curli is made solely by the self-assembly of these CsgA monomers, it presents an excellent target for genetic engineering of a recombinant system. Foreign peptide domains fused onto CsgA at the C-terminus have been shown to be successfully incorporated into the curli structure, allowing for the construction of a biofilm with multiple new appendages simply by altering the CsgA fusion sequence [1]. A mutant strain of E. coli, PQN4, which does not contain the Csg operon responsible for the assembly and secretion of curli nanofibres but does contain an inserted T7 RNA polymerase [5], will be used to create an artificial biofilm. This will ensure that only curli genes inserted on plasmid vectors will be expressed. pETDuet-1 and pET-28a vectors will be used to express recombinant CsgA proteins and the CsgCEFG operon respectively. CsgB, which encodes for the protein unit anchoring CsgA fibres to bacterial cell wall [5], will remain deleted for the purpose of quantifying each type of recombinant CsgA secreted into the surrounding media. CsgD, which encodes for a curli-regulatory system [5], does not contribute to the formation of curli nanofibres and will thus also remain deleted. Both pETDuet-1 and pET-28a, along with their inserts, will be transformed into PQN4 cells. By inserting two different CsgA fusion constructs, CsgA-AFP8 and CsgA-SpyTag, into pETDuet-1, both recombinant CsgA proteins will be exported to the extracellular matrix by the Csg system expressed on pET-28a creating recombinant curli nanofibres. The CsgA-AFP8 fusion will be used to facilitate ice-binding. To append domains that are too large to secrete using the normal Csg pathways, the SpyTag-SpyCatcher system will be used. SpyTag and SpyCatcher form two halves of a split protein system, that covalently bind upon coming into contact with one another [6]. A SpyCatcher fused with C-lectin, a polysaccharide-binding domain from a bacterial adhesin, will be provided exogenously from a pET28b vector. The long adhesin containing the C-lectin domain is endogenously expressed on the surface of Marinobacter hydrocarbonoclasticus [7]. Given that SpyCatcher covalently binds to SpyTag upon contact, the C-lectin fusion protein will adhere the biofilm to Marinobacter hydrocarbonoclasticus by crosslinking via free-floating dextran [8]. M. hydrocarbonoclasticus naturally degrades hydrocarbons, particularly long-chain alkanes [9]. Thus, by genetically engineering PQN4, the abilities to both bind to ice and degrade long-chain hydrocarbons will be provided to the new, bifunctional biofilm. Biofilms are an attractive nanobiotechnological platform given their ability to foster bacterial colonies that behave uniquely relative to their free-living counterparts. Biofilms enhance bacterial survival by enhancing resource capture, facilitating social communication and cooperation, as well as providing a robust physical scaffold to protect against environmental threats. Our genetic engineering of biofilms to incorporate novel functionalities such as ice binding and hydrocarbon degradation allows us to tailor the biofilm matrix to become a living bioremediation factory. Our bifunctional biofilm would be ideally suited for Arctic oil remediation. There is currently no proven effective method for cleaning up an oil spill in icy water. Low temperature, low sunlight, and short productive seasons make natural bioremediation a very slow process in the Arctic. The ice-binding domains of the AFPs expressed on the biofilm would be critical for nucleating large biofilm colonies on Arctic sea ice. Once established, the biofilm could then make use of its other appendage—the hydrocarbon-degrading M. hydrocarbonoclasticus. This bacterium would be ideally located on the extracellular matrix to interact with surrounding hydrocarbons. The catabolites produced by this species would feed into the metabolic pathways of the E. coli, providing additional symbiotic maintenance of the biofilm. Thus, our biofilm could be deployed following an oil spill in Arctic waters to facilitate the degradation of toxic hydrocarbons as a greener approach. This bifunctional biofilm may have many biomedical applications as well. In cystic fibrosis patients, Pseudomonas aeruginosa is among the leading causes of deterioration of their respiratory status. Using a bifunctional biofilm, a non-pathogenic bacterium could recognize and bind to P. aeruginosa and deliver an enzyme that would degrade the extracellular polymeric substances (EPS) of P. aeruginosa. Scientists are already taking advantage of biofilms as protein factories for vaccination development. By creating a bifunctional biofilm, vaccinations could cover more than one strain or pathogen due to multiple antigens being produced.
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