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Team:St Andrews/project/modelling
Modelling
Contents |
Introduction
Hello and welcome to the St. Andrews iGEM 2010 modelling pages. Here we present our work completed throughout the 2010 summer, the methodology behind our various models, and some of the conclusions we drew from those pieces of work.
Components
Our project can be divided into two main components: the engineering of a bistable switch into the LuxR quorum sensing system, and the integration of CqsA (the biosynthetic enzyme present in the cholera system) into the LuxR circuit. The aim of the modeling side of the project was to treat these two tasks independently and on their completion construct a combined model. However, this initial aim was proved to be almost impossible due to the lack of rate constants for the cholera system, which has only been understood in its full complexity relatively recently [1]. In order to reach a compromise, we have built a number of qualitatively accurate models for the bistable LuxR system, and outlined a framework of differential equations for the cholera system which are correct at the time of writing, and which requires more rate constants to be of further use. The work done on the bistable switch included an investigation into why exactly such a configuration of genes exhibits hysteretic behavior, and what parameters are of importance in determining the “level” of bistability of the system.
The LuxR System
The LuxR quorum sensing system is common among the vibrio family, and the lux system of the luminescent bacterium V. Fischeri has been studied in great detail. This gave us a plentiful supply of scientific papers and review articles on which to base our understanding of the system. The LuxR system is underpinned by three quorum sensing molecules, those being the signalling molecule HSL; the HSL synthase LuxI and the transcriptional regulator LuxR. HSL may be generated in the cell by two means, either diffusion through the cell membrane from the external environment or being produced within the cell. Throughout the development of our models we considered a number of ways of trying to replicate this, and the details of these differenent techniques can be found in further sections. Whilst in the cell it may bind to LuxR, and two HSL molecules form a tetramer with two complementary LuxR molecules to form a complex which can then activate the lux operon. In the wild-type, this operon contains the genes coding for LuxI and those ultimately responsible for GFP production, with the luxR gene being located upstream and being constitutively produced. However, in our re-engineered operon the luxR gene lies downstream of the lux operon, thus its expression is also promoted. Hence once the promoter is activated, there is an increased production of LuxI, LuxR and also of GFP, which can therefore be used as an indicator of the promoter activity within the cell. Once transcription has been completed, the LuxI catalyses the production of more HSL from resources within the cell (which we have assumed exist in a sufficiently high concentration that they can be considered infinite). This may leave the cell due to a concentration gradient if the cell density in the medium is low, or otherwise will be used again in the loop, causing a positive feedback effect which will cause a high increase in the concentration of HSL. In this way the single bacterium can communicate with those surrounding it in order to evaluate whether it exists in sufficient numbers to overcome the immune system.]
References
[1] Goryachev, A.B., D.J. Toh and T. Lee, “System analysis of a quorum sensing network: Design constraints imposed by the functional requirements, network topology and kinetic constant.” BioSystems 2006 83, 178-187
[2] Aberdeen 2009, "Parameters" iGEM wiki, <https://2009.igem.org/Team:Aberdeen_Scotland/parameters>
[3] Alon, Uri. “An Introduction to Systems Biology Design Principles of Biological Circiuts.” London: Chapman & Hall/CRC, 2007
[4] Subhayu Basu, “A synthetic multicellular system for programmed pattern formation.” Nature April 2005, 434, 1130-1134
