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Project

Project Overview

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

For details on DspB, please refer to its page.

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 Caulobacter cells can initiate chromosome replication. The swarmer cells differentiate into stalked cells after a period of motility. The stalked cells produce an extremely strong polar adhesive called the holdfast (Evelyn Toh,1 Harry D. Kurtz, Jr.,2 and Yves V. Brun) which can serve as a biofilm initiator. The Caulobacter strain we used (CB15N) is deficient in the gene that is responsible for producing the holdfast.

In the wild, Caulobacter is found in freshwater lakes and streams, where low nutrient 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 many 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 2010). 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 Caulobacter is composed of a single protein—RsaA, which is secreted and assembled into a hexagonal crystalline array that covers the organism. RsaA provides Caulobacter a measure of protection for the cell from attacking agents such as proteases, viruses and parasitic bacteria (p.495). RsaA makes up approximately 10-12% of the total cell protein production. The synthesis of RsaA occurs without need for induction and the protein is produced continuously throughout the life cycle (p. 494). The secretion system for RsaA is also well studied: secretion is ATP-driven and considered a type I secretion pathway given the fact that the C-terminal secretion signal is not cleaved off after secretion (Awram & Smit 1998). The secretion signal has been shown to be within the C-terminal 82 amino acids of RsaA (Bingle, Nomellini and Smit 2000), and proteins as large as RsaA (98kDa, 1025 amino acids (Gilchrist, Fisher and Smit 1992)) can pass through the cell membrane successfully.

Thus, the reasons we chose Caulobacter as our chassis organism can be summarized as follows: 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).

Biofilms

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.

The Experiments

Constructs

Since Caulobacter crescentus has a G/C rich genome (over 60%) and both Esp and DspB coding gene are G/C poor, we obtained the coding gene sequences of both proteins and optimized them to get a better expression in Caulobacter. Gene sequences are designed to maintain the amino acids sequence but make all corresponding DNA codon for the amino acids the most frequently appearing one in Caulobacter’s transcription system.

We had IDT synthesized esp (optimized) and dspB (optimized) for us as the major coding part of our biofilm inhibitor contructs. We do want to see the difference of gene expession level in Caulobacter between the wild type and optimized gene, and make wild type esp gene available for other synthetic biologists who want to use esp gene in organism other than bacteria. Therefore, we acquired wild type esp gene from Staphylococcus epidermidis by PCR. Univeristy of British Columbia Team 2010 was working on DspB protein last year and they have not yet characterized it successfully. We got our wildtype dspB gene from them.

The promoters we were using are Pxyl, PrsaA and BBa_K081005, all functioning at a relatively high efficiency in both E. coli and C. crescentus. Pxyl is a xylose-induced promoter that origins from C. crescentus; PrsaA is a strong constitutive promoter also native to C. crescentus that is responsible for initiating the RsaA protein translation; BBa_K081005 is a strong constitutive promoter that 2008 iGem University of Pavia team designed.

For the RsaA C-terminal secretion tag, since we know from the previous research that as little as 80 C-terminal amino acids can function correctly and a larger secretion signal do increase the yield efficiency of producing heterologous protein, we decided to clone the gene that encodes about 120 C-terminal amino acids from Caulobacter. We attached have engineered the following seven constructs so far:

All the cloning were done with E. coli and after the construct is accomplished, we transferred the construct from pSB1C3 plasmid to pMR 10 plasmid before a mating process that transfer the pMR 10 plasmid with the gene to Caulobacter.

One issue that gave us a hard time in the beginning of the summer was that if we proceed as usual—make standardized esp/dspB and rsaA C-terminal signal with the appropriate prefix and suffix and ligate two parts together—there would be a stop codon in between the two genes that terminates rsaA C-terminal secretion signal expression. Later we solved the issue by modify the suffix of the upstream gene and the prefix of the downstream gene so that the stop codon is not in frame any more.

Part 3

Data & Results

From the biofilm assay and the silver-stained poly-acrylamide gel, we saw significant difference between WT Caulobacter and most of our constructs.