Difference between revisions of "Part:BBa K1682018:Experience"

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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.
 
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.
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===NarL System - Lambert_GA 2020===
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The NarL system is designed based on HKUST-Rice 2015’s part BBa_K1682018, modeling after E.coli’s natural nitrate and nitrite sensor. While Lambert iGEM’s NarL system is modified to change the original design’s terminators due to problematic DNA synthesis in IDT (Integrated DNA Technologies), both HKUST-Rice’s and Lambert’s NarL biosensor detect nitrate [2]. 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.
  
 
===Experience - Lambert_GA 2020===
 
===Experience - Lambert_GA 2020===

Revision as of 23:05, 27 October 2020


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Background - Lambert_GA 2020

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.

NarL System - Lambert_GA 2020

The NarL system is designed based on HKUST-Rice 2015’s part BBa_K1682018, modeling after E.coli’s natural nitrate and nitrite sensor. While Lambert iGEM’s NarL system is modified to change the original design’s terminators due to problematic DNA synthesis in IDT (Integrated DNA Technologies), both HKUST-Rice’s and Lambert’s NarL biosensor detect nitrate [2]. 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.

Experience - Lambert_GA 2020

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, [BBa_B1002 BBa_B1002], after super folder GFP and a rrnBT1 T1 terminator, [BBa_J61048 BBa_J61048], after TetR.

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.

src="T--Lambert_GA--_Nardiagram.png"

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