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Team:Grinnell/Project
From 2011.igem.org
Project
Overall project
Biofilms are cells encased in a hydrated extracellular polymeric substance (EPS) matrix that is composed of polysaccharides, proteins, nucleic acids, and lipids2. Biofilms act as a protective umbrella for their inhabitants against various adverse conditions and can aid in communication between cells3. Biofilms have recently become a concern in various fields, including health, food, and energy. The structure of biofilms make them difficult to remove once mature. By protecting the cells involved and facilitating horizontal gene transfer biofilms increase virulence of the incorporated bacteria.
Synthetic biologists are beginning to tackle the problem of biofilms, as evidenced by the number of iGEM teams interested in the degradation and inhibition of biofilms in recent years. These projects have been conducted using the workhorse of synthetic biology, E. coli, with a focus on finding ways to kill the bacteria in the biofilm before the biofilm is formed (inhibition) or by infiltrating the biofilm (degradation). Our team approached this problem differently in two ways: we aimed to exploit the rigorous type I secretion pathway of Caulobacter crescentus, and we sought to degrade the EPS rather than kill the involved cells.
We decided to utilize Caulobacter because it has many advatages over E. coli for our purposes. The first of these is the rigorous typeI secretion system that Caulobacter uses to secret its paracrystalline S-layer protein, RsaA, which makes up 10-12% of manufactured protein in lab strain CB15N (a strain which is deficient in producing a holdfast). Caulobacter is an aquatic bacterium and it grows well in low-nutrient environments. Like E. coli, Caulobacter is gram-negative and has had its genome sequenced, however Caulobacter is safer for use around humans as it produces 100 times less endotoxin than E. coli, and is unable to survive in a human body. To exploit the secretion pathway, we planned to attach the C-terminal secretion tag from RsaA to a biofilm inhibiting or degrading protein. This allows our system to produce and secrete large quantities of enzyme that are easy to isolate because there is no cell lysis that is necessary.
For the biofilm degrading enzymes that we chose to have Caulobacter secrete, we focused our efforts on a serine protease, Esp, from Staphylococcus epidermidis, and a hydrolase, DspB, from Aggregatibacter actinomycetemcomitans that have both been shown to degrade biofilms8 9.
The general goals of our project were: 1) to introduce Caulobacter as another potential chassis for synthetic biology, especially in environmental and biomedical-related fields; 2) to create a toolbox of biobrick parts that enable easy exploitation of Caulobacter's typeI secretion system for any protein of interest through fusion to the C-terminal secretion tag; 3) and to develop a system for degrading biofilms by targeting the EPS.
Project Details
Caulobacter crecentus and Type I secretion
Caulobacter crescentus is an aquatic gram-negative bacterium. It is widely used as a model organism for studying cell cycle, cell division and cell differentiation. Caulobacter divides asymmetrically to produce two morphologically different progeny, a swarmer and a stalked daughter cell (Laub Shapiro McAdams). Only the stalked cell stage Caulobacter can initiate chromosome replication and the swamer cell will later differentiate into stalked cell after a period of motility. The stalked cell can produce an extremely strong polar adhesin called the holdfast (Evelyn Toh,1 Harry D. Kurtz, Jr.,2 and Yves V. Brun) and therefore can serve as a biofilm initiator. The Caulobacter strain we used in the project is CB15N, which is deprived of the gene that is responsible for producing holdfast.
In the wild, C. crescentus is found in freshwater lakes and streams, where extended periods with low nutrients conditions are common. However, Caulobacter cells are well equipped with various environmental sensors and transporters so that they are able to survive. For example, Caulobacter has way more TonB-dependent receptors than most bacteria, which is accountable for gathering carbohydrates from a variety of sources (Kathleen R. Ryan, James A. Taylor3 and Lisa M. Bowers 20010). Sometimes Caulobacter is even capable of halting cell cycle progression in extreme dilute aquatic environment. (Laub Shapiro McAdams)
One feature of Caulobacter that is often overlooked is its surface layer. The S-layer of the Caulobacter is omposed of a single protein—RsaA, which is secreted and assembled into a hexagonal crystalline array that covers the organism. RsaA provide Caulobacter a measure of protection for the cell from attacking agents such as proteases, viruses and parasitic bacteria (p.495). The RsaA protein makes up a large proportion of proteins that the cell makes, approximately 10-12% of the total cell protein. The synthesis of RsaA occurs without need for induction and the protein is produced continuously throughout the life cycle (p. 494). The secretion signal of the Caulobacter is also well studied. RsaA secretion is ATP-driven and considered as a type I secretion given the fact that the C-terminal secretion signal is not cleaved off after the process (Awram & Smit 1998). The secretion signal has been shown to be within the C-terminal 82 amino acids of the molecule in RsaA gene (Bingle, Nomellini and Smit 2000), and protein as large as RsaA (98 kDa protein, consists of 1025 amino acids (Gilchrist, Fisher and Smit 1992) can pass through the cell membrane successfully.
Thus, the reason we chose Caulobacter crescentus specifically as the final carrier of our biofilm inhibiting machine is well summarized as followed: Caulobacters secrete recombinant proteins using the Type I secretion system, not the General Secretory Pathway (GSP), which is more common. The bacteria are obligate aerobes and grow to high densities in minimal media, up to OD 25-30. They do not secret other proteins and have a lipopolysaccharide (LPS) that has surprisingly low endotoxin potential. They are easily manipulated in lab and have a sequenced genome. And the yields are usually high for heterologous protein secretion (p.484).
Biofilm
Biofilms are a unique community of usually heterogeneous microorganisms that readily attach to rough and hydrophobic surfaces1. They are composed of cells encased in a hydrated extracellular polymeric substance (EPS) matrix that is composed of polysaccharides, proteins, nucleic acids, and lipids2.
Due to its extracellular structure, a biofilm can act as a protective barrier of its dwellers against various adverse environments and can aid in the communication between cells3. Cells within a biofilm are frequently found to be more resistant to antimicrobials compared to planktonic cells4. Moreover, EPS serves as a bridge among cells, promoting intercellular communication that may facilitate cells’ (or biofilm as a whole) adaption to changing environment5.
These traits make the elimination of biofilms a challenging concern in a wide range of areas, especially in food industry, environmental and biomedical areas1. Pathogens, such as Escherichia coli O157:H7 and Staphylococcus aureus, may contaminate the food products, microorganisms in biofilms catalyze chemical and biological reactions causing metal corrosion in pipelines and tanks, and they can reduce heat transfer efficacy if the biofilm becomes too thick1 6.
