Signalling

Part:BBa_K3411020

Designed by: Janet Standeven, Nikhita Lalwani, Sindhura Mettupalli, Kathy Ye, Sachintha Ashok, Saif Khan   Group: iGEM20_Lambert_GA   (2020-10-22)
Revision as of 23:44, 27 October 2020 by Saifkhan (Talk | contribs) (→‎NarP Nitrate/Nitrite Biosensor Design)


NarP Nitrate/Nitrite Sensor

Native Nar Operon

Figure 1: Diagram of the Pho Regulon signaling pathway. The Pho Regulon responds to extracellular Pi 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.

NarP Nitrate/Nitrite Biosensor Design

Figure 2: Diagram of the Nitrate detection NarP system construct.
Figure 3: Diagram of the Nitrate detection NarL system signalling transduction pathway.


Lambert_GA iGEM 2020's novel NarP system, BBa_K3411020, is also modeled after E.coli’s natural nitrite sensor in the NAR Operon. Because the NarL system (BBa_K1682018) detects nitrate, Lambert iGEM needed a method to detect levels of nitrite: NarP, which detects both nitrate and nitrite. By calculating the corrected difference, the system would have concentrations for both nutrients. Lambert iGEM’s novel NarP composite part BBa_K3411020 and the NarP gene BBa_K3411010 are also modeled after E.coli’s natural nitrite sensor. Similarly to NarL, the promoter BBa_J23106, a constitutive Anderson Promoter, produces TetR, which represses the PtetO promoter, producing NarP. By regulating the amount of aTc, the molecule that inhibits TetR, the lab can control NarP’s expression, the product of the PtetO promoter. NarQ, the native membrane-bound protein, senses the amount of nitrate and/or nitrite in the cell: in the presence of nitrite and/or nitrate, NarQ will phosphorylate NarP, activating it, but if there is no nitrite and/or nitrate present, NarQ will not phosphorylate NarP [1]. If NarP is phosphorylated, it will activate the nirB promoter, producing GFP [2]. Lambert iGEM plans to correlate GFP levels to nitrite through mathematical modeling.

Lambert_GA iGEM 2020's plans on cloning and characterizing our NarP composite part in the coming year as a part of our two-year project, AgroSense.

References

[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] Wang H., & Gunsalus R. (2000). The nrfA and nirB Nitrite Reductase Operons in Escherichia coli Are Expressed Differently in Response to Nitrate than to Nitrite. Journal of Bacteriology, 182(20): 5813–5822. doi: 10.1128/jb.182.20.5813-5822.2000


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2000
    Illegal NheI site found at 2023
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2727
    Illegal AgeI site found at 1371
  • 1000
    COMPATIBLE WITH RFC[1000]


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