Team:Wageningen UR/Project/IntroductionProj1

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Building a Synchronized Oscillatory System

The Synchroscillator: a Synchronized Oscillatory System

Abstract

One aim of synthetic biology is to re-engineer naturally occurring gene circuits to produce artificial systems that behave predictably. Our project involved providing additional functionality and also streamlining a recently published synchronized oscillatory circuit in an attempt to reproduce and further characterize its dynamics. Our genetic circuit consists of modified (and BioBricked) elements of the Vibrio fischeri lux quorum sensing system composed to form interconnected positive and negative feedback loops, which dynamically regulate the expression of GFP. In order to provide our E. coli host with the right environment required for population-wide oscillations, we designed and manufactured a custom flow-chamber capable of maintaining a defined cell population while independently varying the growth conditions. The chamber was specifically designed for time-lapse studies with a fluorescence microscope. We detected synchronized oscillatory gene expression under zero-flow conditions, suggesting an unexpected level of robustness. This should facilitate its integration with more advanced genetic circuits.

Fig. 1 Olympus microscope exciting GFP in the flow chamber, the Laboratory of Microbiology, Wageningen

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-== Synchronized Oscillatory System ==
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-The aim of this project is to design and implement a system exhibiting sustained oscillatory protein expression which should be visible and synchronized on the scale of a physically constrained population of E. coli cells. The principles that govern this type of behaviour have been studied both in theory and in practice, and as such there exists a solid foundation to apply these ideas in the context of the iGEM competition. In essence, this project consists of constructing a plasmid containing genes, the products of which reciprocally affect each other’s expression in a reliable manner. Based on a previously established design, we intend to take advantage of the great variety of standardized, interchangeable and freely available Bio–Bricks to construct modified genetic circuits, aiming at an improved bacterial oscillator. Due to the difficulty in experimentally verifying the phenomena we wish to observe, special considerations regarding the experimental set-up will have to be made.
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+== The Synchroscillator: a Synchronized Oscillatory System ==
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-The starting point for the genetic circuitry we intend to make is a design recently published in the article “a synchronized quorum of genetic clocks” by Danino & Hasty. This genetic circuit uses natural elements of bacterial quorum sensing systems to form coupled positive and negative feedback loops which control the expression of a reporter protein: the LuxI enzyme that catalyses the last step of the acyl-homoserine lactone (AHL) biosynthesis, the AHL-responsive transcriptional regulator LuxR, and the reporter Green Fluorescent Protein (GFP) (Fig.1). The AHL molecules can easily diffuse through cell membranes to the extracellular medium. This allows all the cells in a culture to influence each other’s activity in a uniform manner (quorum sensing). The result being that the oscillations arising from the genetic feedback loops are synchronized on the scale of a whole cell culture. All of the parts used in this design exist in the Registry of Standard Biological Parts and are freely available for our use. [[Team:Wageningen_UR/Project/CompleteProject1Description| Read more]]
+=== Abstract ===
+One aim of synthetic biology is to re-engineer naturally occurring gene circuits to produce artificial systems that behave predictably. Our project involved providing additional functionality and also streamlining a recently published synchronized oscillatory circuit in an attempt to reproduce and further characterize its dynamics. Our genetic circuit consists of modified (and BioBricked) elements of the ''Vibrio fischeri'' lux quorum sensing system composed to form interconnected positive and negative feedback loops, which dynamically regulate the expression of GFP. In order to provide our ''E. coli'' host with the right environment required for population-wide oscillations, we designed and manufactured a custom [[Team:Wageningen_UR/Project/Devices| flow-chamber]] capable of maintaining a defined cell population while independently varying the growth conditions. The chamber was specifically designed for time-lapse studies with a fluorescence microscope. We detected synchronized oscillatory gene expression under zero-flow conditions, suggesting an unexpected level of robustness. This should facilitate its integration with more advanced genetic circuits.
+[[Team:Wageningen_UR/Project/CompleteProject1Description| Read more]]
-[[File:SOS_2.png|center]]
+[[File:Measuring_GFP_WUR.jpg|600px|center]]
+'''Fig. 1''' ''Olympus microscope exciting GFP in the flow chamber, the Laboratory of Microbiology, Wageningen''
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