Part:BBa_K1682018:Experience

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Native Nar Operon - Lambert_GA 2020

Figure 1: Diagram of the Pho Regulon signaling pathway. The Pho Regulon responds to extracellular P_i levels and transcribes its regulatory genes.

Native to E. coli, the Nar Operon regulates anaerobic gene expression in response to two electron acceptors: nitrate and nitrite. This system consists of two homologous membrane-bound sensor proteins (NarX and NarQ) as well as two homologous DNA-binding response regulators (NarL and NarP). NarL’s conjugate is the membrane-bound NarX protein while NarP’s conjugate is the membrane-bound NarQ protein. Lambert iGEM is utilizing this system to test nitrate and nitrite levels, NarL and NarP expression, and downstream GFP expression through mathematical models.

NarL System - Lambert_GA 2020

Figure 2: Diagram of the Nitrate detection NarL system construct based on HKUST-Rice 2015 Part:BBa_K1682018.

The NarL system is designed based on HKUST-Rice 2015’s part BBa_K1682018, modeling after E.coli’s natural nitrate and nitrite sensor. Lambert iGEM’s NarL system is modified to change the original HKUST-Rice 2015 design’s terminators due to problematic DNA synthesis. To optimize functionality, Lambert_GA altered the original part by replacing the original terminators with rrNB T1 terminator and T7Te terminator, BBa_B1002, after super folder GFP and a rrnBT1 T1 terminator, BBa_J61048, after TetR. Both HKUST-Rice’s and Lambert’s NarL biosensor detect nitrate [2].

Figure 3: Diagram of the Nitrate detection NarL system signalling transduction pathway.

The promoter BBa_J23106, an Anderson Promoter, constitutively produces TetR, which represses the PtetO promoter that produces NarL. By regulating the amount of aTc, the molecule that inhibits TetR, the team can control levels of NarL. NarX, the native membrane-bound protein, senses the amount of nitrate in the cell: in the presence of nitrate, NarX will phosphorylate NarL, activating it; however, if there is no nitrate present, NarX will not phosphorylate NarL [1]. The PdcuS promoter, which naturally produces green fluorescent protein (GFP), is repressed by phosphorylated NarL which causes lower GFP levels in the presence of nitrate [3]. Lambert iGEM plans to correlate the GFP levels to nitrate through mathematical modeling.

Applications of BBa_K1682018

Lambert_GA 2020 is working on demonstrating that this part can be transformed into competent E.coli cells while retaining functionality. Attempts at ligation both with restriction digest cloning and Gibson Assembly cloning failed. Our team is continuing to troubleshoot and is still in the cloning process. In 2021, we plan on utilizing this to model a nitrate biosensor in hydroponic/aquaponic systems.

References - Lambert_GA 2020

[1] Darwin A.J., & Stewart V. (1996) The NAR Modulon Systems: Nitrate and Nitrite Regulation of Anaerobic Gene Expression. In: Regulation of Gene Expression in Escherichia coli. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-8601-8_17

[2] HKUST-Rice iGEM (2015). Potassium, Phosphate and Nitrate Biosensors. Retrieved from http://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR

[3] Gob E., Bledsoe P., Chen L., Gyaneshwar P., Stewart V., & Igo M. (2005). Hierarchical Control of Anaerobic Gene Expression in Escherichia coli K-12: the Nitrate-Responsive NarX-NarL Regulatory System Represses Synthesis of the Fumarate-Responsive DcuS-DcuR Regulatory System. Journal of Bacteriology, 187(14): 4890–4899. doi: 10.1128/JB.187.14.4890–4899.2005

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