Team:Lambert GA/Parts
Lambert_GA iGEM 2020's NarL system is modeled after E.coli’s natural nitrate sensor, the Nar Operon, which includes includes NarL, the response regulator, and NarX, the membrane-bound sensor protein. The NarL/NarX system detects nitrate. This specific part is the isolated NarL gene.
Lambert_GA iGEM 2020 will continue cloning and begin characterization of this NarL part in the coming year as a part of its two-year project, AgroSENSE.
The NarL gene is a basic, signalling part created by Lambert_GA 2020. Due to a change in iGEM parts registry requirements, the HKUST-Rice 2015 team did not upload the basic part, however they used the gene in their BBa_K1682018 composite part. (See: Wetlab: Nar)
The NarP gene is a basic, signaling part created by Lambert_GA 2020 and is within the novel BBa_K3411020 NarP composite nitrate/nitrite biosensor.
Lambert_GA iGEM 2020's NarP system is modeled after E.coli’s natural nitrite and nitrate sensor: the Nar Operon, which includes the NarP response regulator, the gene listed above, and NarQ, the membrane-bound sensor protein. The NarP/NarQ system detects both nitrite and nitrate. This specific part is the isolated NarP gene.
Lambert_GA iGEM 2020 plans on cloning and characterizing this NarP part in the coming year as a part of its two-year project, AgroSENSE. (See: Wetlab: Nar)
Figure 1. Construct of NarL composite part.
HKUST-Rice 2015 created this composite part as a nitrate biosensor utilizing the NarX/NarL Two-Component Signal Transduction System in E. coli.
This year, Lambert_GA is working on demonstrating that this part can be transformed into competent E.coli cells while retaining functionality. Cloning has been unsuccesful so far with both restriction digest and gibson assembly into pSB1C3.
To optimize functionality, Lambert_GA altered the original part designed by HKUST-Rice 2015 by replacing the original terminators with rrNB T1 terminator and T7Te terminator after super folder GFP and a rrnBT1 T1 terminator after TetR. (See: Wetlab: Nar)
Lambert_GA iGEM 2020's novel NarP system, BBa_K3411020, is modeled after E.coli’s natural nitrite sensor in the Nar Operon, specifically NarQ/NarP.
Lambert_GA iGEM 2020's plans on cloning and characterizing this NarP composite part in the coming year as a part of its two-year project, AgroSENSE. (See: Wetlab: Nar)
To analyze the amount of nitrate, Lambert_GA iGEM 2020 needed to correlate nitrate levels to a reporter protein. Thus, the team needed a promoter to respond to phosphorylated NarL and report downstream GFP, a well-characterized fluorescent protein. HKUST-Rice used the PdcuS protomer, which is supported by an article in the NCBI concerning potential promoters’ sensitivity to phosphorylated NarL levels. Phosphorylated NarL then represses the PdcuS promoter, lowering GFP levels in the presence of high levels of nitrate.
The PdcuS promoter is a basic, regulatory part created by Lambert_GA 2020. Due to a change in iGEM parts registry requirements, the HKUST-Rice 2015 team did not upload the basic part, even though the team used the promoter in their BBa_K1682018 composite part. (See: Wetlab: Nar)
This composite part, created by NUS_Singapore 2017 is an extracellular phosphate sensor with GFP reporter based on part BBa_K116404 (NYMU Taipei 2008). Lambert_GA 2020 characterised this part by creating characterization curves using fluorescence data and a deterministic ODE model.
Part BBa_K2447000 utilizes the Pho Regulon - replacing the Pho Regulon genes with Green Fluorescence Protein (GFP). Similar to the signaling pathway, under high extracellular phosphate levels, activation of the promoter via binding of phosphorylated PhoB transcription factor results in the downstream transcription and expression of GFP.
Lambert_GA 2020 used this part as a biosensor for hydroponics/aquaponics systems as research suggested the sensitivity range of the part is within the optimal range of phosphate concentration for hydroponics/aquaponics systems: 50uM to 100uM. To improve upon the existing characterization of the phosphate sensor, Lambert iGEM tested with a greater number of phosphate concentrations ranging from 0uM to 100uM in intervals of 20uM. (See: Wetlab: Pho)