Composite

Part:BBa_K3725220

Designed by: Adam Silverman   Group: iGEM21_Lambert_GA   (2021-10-19)
Revision as of 17:23, 20 October 2021 by Saifkhan (Talk | contribs) (→‎Design)


Phosphorylated NarL-Specific w/ GFP Reporter

Lambert_GA iGEM 2021’s nitrate biosensor is modeled after E.coli’s natural nitrate sensor, the Nar Operon, which includes the membrane-bound sensor protein NarX and its corresponding DNA-binding response regulator NarL. The NarL/NarX system detects nitrate by using phosphorylated NarL as an inducer of promoter part BBa_K3725210 (Promoter pNar). In the presence of nitrate, NarX phosphorylates NarL, which activates pNar, and sfGFP is transcribed. Conversely, in the absence of nitrate, NarX does not phosphorylate NarL and sfGFP is not transcribed. To analyze the concentration of nitrate present in a solution, nitrate levels need to be correlated with a reporter protein, or a promoter responsive to phosphorylated NarL and report downstream GFP, a well-characterized fluorescent protein.

Part Design

Figure 1. The diagram above shows the construct of Part BBa_K3725220.

This plasmid was shared by Dr. Adam Silverman from Jewett Lab at Northwestern University.

Usage and Biology

Figure 1. The diagram above shows NarX (the native E. coli Nar membrane-bound sensor protein) and NarL (the corresponding DNA-binding response regulators), as well as how it works with our pNar Composite Part.

Lambert_GA iGEM 2021’s nitrate biosensor is modeled after E.coli’s natural nitrate sensor, the Nar Operon, which includes the membrane-bound sensor protein NarX and its corresponding DNA-binding response regulator NarL. The NarL/NarX system detects nitrate by using phosphorylated NarL as an inducer of promoter part BBa_K3725210 (Promoter pNar). In the presence of nitrate, NarX phosphorylates NarL, which activates pNar, and sfGFP is transcribed. Conversely, in the absence of nitrate, NarX does not phosphorylate NarL and sfGFP is not transcribed. To analyze the concentration of nitrate present in a solution, nitrate levels need to be correlated with a reporter protein, or a promoter responsive to phosphorylated NarL and report downstream GFP, a well-characterized fluorescent protein.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 930
    Illegal BsaI site found at 1108
    Illegal SapI.rc site found at 133


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Parameters
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