Team:Paris Bettencourt/Logic Circuit

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@iGEM_Paris

LOGIC CIRCUIT

Introduction & Background

For our main system to form, where production of a biomaterial, only occurs when two specific lights intersect, a logic-gate needed to be developed. To create a NOR-gate at the promoter level, we aimed at creating dually repressed promoters. Most of NOR-gate promoter designs using tandem repressible promoters (1, 2, 3) have unpredictable properties and leaky expression. The main challenge to create a clean design of NOR-gate containing only one transcription starting point is the lack of standard transcriptional elements smaller than repressible promoters. Recent work on transcription elements showed that assembling insulated synthetic operator upstream and downstream of an insulated T7 promoter core allowed for a more diverse control of gene expression and a more specific response time (1).

Design

Based on our modeling results, we designed decided to work of three specific repressors due to their interesting parameter values. For each pair of repressors, four different arrangements of the operators were characterized experimental. Firstly, the impact of the way the two operators are ordered downstream of the promoter core was studied (Figure 2A). Secondly, we studied the impact of adding a second operator upstream of the promoter core on promoter activity (Figure 2B). This gave us a total of twelve promoters to test (Table 1). Different input combinations were applied to the system, i.e. repressor concentrations were varied. In order to control concentration, the repressors were put under the control of well-known inducible promoters : p_lacI and p_ara. Since it is difficult to track the concentration of each repressor in real time, it was approximated by the fluorescence emitted by a fluorescent protein - eyfp or ecfp - regulated by the same inducible promoters as the repressors. Output was measured by the mRFP1 fluorescence emitted. The aforementioned florescences were measured by flow cytometry. (Figure 2)

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com