Team:Calgary

The iGEM competition is a synthetic biology competition held annually at Massachusetts Institute of Technology (MIT) in November. Started in 2004, the iGEM Jamboree brings together teams from multiple universities to compete on an international stage. The competition allows undergraduate teams from all over the world to be part of the emerging field of Synthetic Biology, an intertwining of principles from Biology and Engineering. For more information about iGEM, please visit their official website.

The U of C iGEM team is made up of 15 undergraduate students from Health Sciences, Engineering, Computer Science and Kinesiology as well as 4 facilitators. For more information regarding the U of C iGEM Enterprise, please visit our website.

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	Communication is essential in our everyday lives; it drives collaboration, understanding, and progress. The University of Calgary iGEM Team recognizes the importance of communication in developing the field of synthetic biology. This year, our team is exploring the development of a communication system in E. coli: the AI-2 signalling system. This system allows for the coordination of bacterial behaviour, essentially allowing a large group to act as a single organism.
	Using mathematical modeling, we are developing models that allow us to predict and evaluate the signalling system's behaviour, thus characterizing the circuit. We have chosen to approach modelling of our system through membrane computing and Matlab.
	Beyond the development of a communication system between bacteria, we have also explored and demonstrated methods of communication with the public. For synthetic biology to flourish, there needs to be meaningful discourse between individuals, whether within the synthetic biology community, with other disciplines, or with the public. This is especially important when discussing the ethical issues that arise from synthetic biology. Our team is examining multiple aspects of concerns regarding synthetic biology, and looking at novel, interesting ways to communicate the results of our exploration to the public.
	For meaningful discourse to occur, all participants require a working knowledge of the concepts they are discussing. Education is a fundamental aspect of providing and spreading such information. This year, our team is using multiple approaches in spreading information to the public regarding iGEM and synthetic biology, as well as providing educational opportunity to students. Such outreach programs include our High-School workshop. We are also developing an educational tool for teaching synthetic biology within the virtual program Second Life. In Second Life, we are creating the means to teach the potentials of synthetic biology, the assembly of genetic circuits, and molecular biology lab procedures. We hope that our virtual learning environment will allow for the training of future iGEMers in molecular biology.
	In addition to utilizing education and outreach to raise the profile of synthetic biology, our fundraising endeavors have also allowed us to create more interest in this field. Our communication with businesses in many industrial sectors allows for the discussion of the opportunities and future developments provided by synthetic biology and the potential applications of our signalling circuit.
	Another important aspect of communication in this competition is the availability of collaborative work, within our own team and as a community itself. Our team is made up of students from health sciences, kinesiology, and engineering; who are actively participating in interdisciplinary work. This requires the ability to communicate information to people from different backgrounds and perspectives. As well, iGEM allows us to communicate with other teams at an international scale. We hope to establish means of communication and thus cooperation with other teams in our respective projects, enhancing the iGEM experience. The wetlab, modelling, second life, human practices, and fundraising components of our project have allowed us to establish communication at the cell, individual, and synthetic bio community levels.

Bacteria are able to communicate by producing and releasing chemical signal molecules termed autoinducers in a process called Quorum Sensing (QS) (1). An increase in local population density of bacteria results in the accumulation of autoinducers until a minimal threshold concentration is reached, whereby bacteria are able to organize their behaviour by coordinating their gene expression. Such coordinated behaviour includes virulence induction, swarming, biofilm formation and genetic competence (2).

QS was first observed in the bioluminescent bacteria Vibrio fischeri (3), where light was emitted only at high population densities, but could be induced in low population densities with the presence of an extracellular substance, later identified as the autoinducer N-acylhomoserine (AHL) (4).

Further research in QS led to the discovery of the universal signaling molecule (5) autoinducer-2 (AI-2), which has been characterized in the gram-negative, bioluminescent marine bacterium Vibrio harveyi (1). AI-2 binds to the periplasmic protein LuxP forming an AI-2-LuxP complex that interacts with LuxQ, a membrane bound histidine kinase (6). At low population density corresponding to low AI-2 levels, this AI-2 signalling acts as a phosphorylation cascade, resulting in the phosphorylated form of luxO. Phospho-LuxO complexes with transcription factor σ54 to activate the transcription of the genes encoding five regulatory small RNAs (sRNAs) termed Qrr1-5 (7). These sRNAs bind and destabilize the mRNA of luxR (8) , a transcriptional activator of the luciferase operon luxCDABE (9). As the mRNA of luxR is degraded in the presence of low levels of AI-2 and low cell density, V. harveyi will not express bioluminescence.

In high population densities and thus high AI-2 levels, LuxQ changes from a kinase to a phosphotase, and the result is unphosphorylated LuxO (1). There is no complexing with σ54, and no production of sRNAs. This leads to unblocked luxR mRNA allowing its translation that drives the expression of bioluminescence via luciferase.

We, the University of Calgary's 2009 iGEM team, have engineered this Vibrio harveyi AI-2 signaling system in Escherichia coli using the molecular cloning techniques used in the International Genetically Engineered Machines (iGEM) competition. This system is coupled with the expression of aiiA, a gene that encodes an AHL-degrading enzyme partaking in quorum quenching, allowing us to target biofilm maintenance.

References
(1)Waters, C.M. & Bassler, B.L.. Quorum sensing: cell-to-cell communication in bacteria. Annu. Rev. Cell Dev. Biol. 21, 319-346 (2005).
(2)Hardman, A.M., Stewart, G.S. & Williams P. Quorum sensing and the cell-cell communication dependent regulation of gene expression in pathogenic and non-pathogenic bacteria. Antonie van Leeuwenhoek. 74, 199-210 (1998).
(3)Nealson, K. H., Platt, T. & Hastings, W. Cellular Control of the synthesis and activity of the bacterial bioluminescent system. J. Bacteriol. 104, 313-322 (1970).
(4)Eberhard, A., Burlingame, A.L., Kenyon, G.L., Nealson, K.H. & Oppenheimer, N.J. Structural identification of autoinducer of Photobacterium fischeri luciferase. Biochemistry. 20, 2444-2449 (1981).
(5)Sun, J., Daniel, R., Wagner-Dobler I. & Zeng, A.P. Is autoinducer-2 a universal signal for interspecies communication: a comparative genomic and phylogenetic analysis of the synthesis and signal transduction pathways, BMC Evol. Biol. 4, 36 (2004).
(6)Bassler, B.L., Wright, M., Silverman, M.R. Multiple signaling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol. Microbiol. 13, 273-286 (1994).
(7)Lilley, B.N. & Bassler, B.L. Regulation of quorum sensing in Vibrio harveyi by LuxO and sigma-54. Mol. Microbiol. 36, 940–54 (2000).
(8)Lenz, D.H., Mok, K.C., Lilley, B.N., Kulkarni, R.V., Wingreen, N.S. & Bassler, B.L.The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholerae. Cell 118, 69–82 (2004)
(9)Swartzman, E., Silverman, M. & Meighen, E.A. The luxR gene product of Vibrio harveyi is a transcriptional activator of the lux promoter. J. Bacteriol. 174, 7490–7493 (1992)