Team:Wageningen UR/Project/IntroductionProj1

Abstract

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 (REF), we intend to take advantage of the great variety of standardized, interchangeable and freely available Bio–Bricks (genes and regulatory elements; REF iGEM website) 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.

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 (REF). 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 LucR, 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 (REF). Read more