Team:Paris Bettencourt/Logic Circuit

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@iGEM_Paris

LOGIC CIRCUIT

Introduction & Background

For our main system to work, 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. The output was measured by the mRFP1 fluorescence emitted. All florescences were measured using a flow cytometer.

Figure 1: Design of the system to test dually repressible promoters. Each promoter is composed of at least two different operators (in blue) recognized by specific repressors and regulates the expression of a reporter gene (mRFP1 in red). A - Only one copy of each repressor is present downstream of the promoter core. B - A copy of one of the two different operators is present upstream of the promoter core to possibly enable a better control of gene expression.

Figure 2: Design of the repressor reporter system. A - System for P_LacI. B - System for p_Bad.

Part number	Part Name	Sequence	Size
BBa_K2510018	T7 - O-HKcI - O-P22c2 promoter	GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGATTTAAGTGTTCTTTAATCGCTGTTCCGCTG	74
BBa_K2510019	T7 - O-HKcI - O-TetR promoter	GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGTCCCTATCAGTGATAGAGATCACACTCCTTC	74
BBa_K2510020	T7 - O-P22c2 - O-HKcI promoter	GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTGAACCATAAGTTCAGCTCTG	74
BBa_K2510021	T7 - O-P22c2 - O-TetR promoter	GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTCCCTATCAGTGATAGAGATCACACTCCTTC	84
BBa_K2510022	T7- O-TetR - O-HKcI promoter	GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCTGAACCATAAGTTCAGCTCTG	74
BBa_K2510023	T7 - O-TetR - O-P22c2 promoter	GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG	84
BBa_K2510024	O-HKcI - T7 - O-HKcI - O-P22c2 promoter	GTGAACCATAAGTTCAGCTATGTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGATTTAAGTGTTCTTTAATCG CTGTTCCGCTG	95
BBa_K2510025	O-HKcI - T7 - O-HKcI - O-TetR promoter	GTGAACCATAAGTTCAGCTATGTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGTCCCTATCAGTGATAGAGAT CACACTCCTTC	95
BBa_K2510026	O-P22C2 - T7 - O-P22C2 - O-HKcI promoter	GTCATTTAAGTGTTCTTTAATGAGCATCTGCTATGTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTGAACCATAAGTTCAGCTCTG	107
BBa_K2510027	O-P22c2 - T7 - O-P22C2 - O-TetR promoter	GTCATTTAAGTGTTCTTTAATGAGCATCTGCTATGTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCG CTGTCCCTATCAGTGATAGAGATCACACTCCTTC	118
BBa_K2510028	O-TetR - T7 - O-TetR - O-HKcI promoter	GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCTGAACCATAAGTTCAGCTCTG	115
BBa_K2510029	O-TetR - T7 - O-TetR - O-P22C2 promoter	GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG	125

Table 1: Designed promoters

Experimental results

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