Team:UNC-Chapel Hill/Background

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BACKGROUND

Project Overview Our team aims to create a novel sensing device that enables one to quantitatively characterize the concentration, or presence of certain compounds. The theoretical advantages to our design include; a wider range of sensitivity, better accuracy, customizability, and ability to detect more than one input at any given time. Our design combines three promoters upstream of three different chromoproteins onto one plasmid. This allows for the advantages listed above (see Figure 1-label theoretical comparison- comparison between tricolor and single color). To provide an example of our general design, we decided to create a tri-color system for glucose. The first two promoters in our system are already apart of the iGEM registry. The first promoter is part BBa_K118011, submitted to iGEM in 2008 by University of Endinburgh in Scotland. This promoter is similar to the familiar promoter for the lactose operon in E. coli cells (the same one as described above). However, BBa_K118011 only has a lone cAMP receptor protein (CRP) binding site, as opposed to having a CRP binding site and an operator site that binds to the lactose repressor protein. At extremely low glucose concentrations cAMP levels in the cell are high. In these conditions, cAMP associates with CRP forming a complex that binds to the binding site and activates high levels of transcription. In our construct, downstream of this glucose repressible promoter is efor Red (BBa_K1073023), which contains its own ribosomal binding site (RBS). The next promoter was also submitted previously by another team. In 2012, Wuhan University in China submitted part BBa_K861171, which is a glucose inducible promoter. The mechanism for this promoter can be thought of as the opposite of the first promoter. Instead of having a CRP binding site upstream of the promoter, the CRP binding site is located within the binding site for DNA polymerase. Thus, has the opposite effect as the first; when glucose is low, cAMP levels are high. Thus, cAMP associates with CRP and blocks transcription. As glucose concentrations rise, transcription is induced. In our construct, the chromoprotein associated with the second promoter is aeBlue (BBa_K1073021). There was not another glucose inducible promoter in the iGEM regresistry, so our team created a novel promoter. This third promoter takes advantage of a repressor protein known as Mlc, which is discussed below. The third promoter in our design is upstream of a yellow chromoprotein. At extremely low concentrations of glucose, the action of the most sensitive promoter dominates, which is our device is always a repressible promoter upstream of a red chromoprotein. Thus, at low concentrations (of the input) the solution appears red. However, when concentrations increase, the red color diminishes and the second promoter starts becoming activated. This produces a blue color as the result of transcription of a blue chromoprotein. Lastly, the third promoter will activate at even higher concentrations resulting in the transcription of a yellow chromoprotein. At these high concentrations the blue and yellow colors will mix and appear green. This unique tri-color system allows for the quantitative measurement of concentrations because they are associate with visible colors. MLC (Makes Large Colonies) Our iGEM project aims to introduce a novel glucose sensing system in which glucose-responsive promoters drive the expression of three reporter chromoproteins. Using inspiration from a previous iGEM team, we designed four novel glucose-inducible promoters called MLC’s (makes large colonies). MLC is encoded by the gene dgsA, and is a repressor regulator of many phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTSs). These are pathways for carbohydrate uptake (glucose). Mlc binds directly to palindromic DNA sequences and blocks RNA polymerase from proceeding with transcription. In E. coli, Mlc specifically regulates the gene pstG, which encodes for the transmembrane glucose permease also called enzyme IICBGlu (regulation also involves a CRP binding site). The protein Mlc has high intracellular concentration when glucose concentration is low, and as a result, a promoter with Mlc binding sites would function as a glucose inducible promoter..

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