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. General Background on MLC (Makes Large Colonies) The third promoter created and was novel promoter to the iGEM parts registry, consisting of Mlc binding sites. Mlc is a regulatory protein that stands for “Making Large Colonies”, and is encoded by the gene dgsA. Specifically, Mlc is a repressor regulator of many phosphoenolpyruvate-dependent carbohydrate phosphotransferase systems (PTSs). These are pathways for carbohydrate uptake (including glucose). Mlc binds directly to palindromic DNA sequences and blocks RNA polymerase from proceeding with transcription.1,2 In E. coli, Mlc is involved in the regulation of many genes involving PTS, including the gene pstG, which encodes for the transmembrane glucose permease also called enzyme IICBGlu (regulation of pstG also involves a CRP, which activates transcription in the presence of glucose: it is thought that the two counter balance one another to provide the correct levels of transcription specifically for enzyme IICBGlu). The protein Mlc has high intracellular concentration when glucose concentration is low and becomes sequestered to the cell membrane when glucose concentrations rise. As a result, a promoter with Mlc binding sites should function as a glucose inducible promoter (repressing transcription at low glucose concentrations). For our project, we synthesized four variants of the Mlc promoter. The promoters vary in two characteristics: the strength of the promoter associated with the Mlc region and the placement of the Mlc region. Using the iGEM parts registry, the two promoters selected were BBa_J23112 (weak) and BBa_J23100 (strong) and one Mlc binding region was placed either prior or after (post) the promoter (strong and weak refer to the relative frequency to which the promoters actually transcription, with strong being more frequent than weak). The appropriate BioBrick restriction sites will also be included (prefixes and suffixes so that 3A assembly protocol can be used for the Mlc promoters). Also primer sequences were synthesized around the promoters so that the promoter could undergo PCR. The sequences for the Mlc promoters and the primer are shown below (Figure 2).

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