Team:HKUST-Rice/Nitrate Sensor PdcuS

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Nitrate Sensor - PdcuS

Phosphate sensor

Nitrate sensor (yeaRp)

Nitrate as a Macro-nutrient

1. Nitrate is an essential macronutrient in plants
2. Probably Wikipedia has some nice stuff about how it affects plant growth/healthy development, etc.
3. It is important that soil has nitrate to ensure healthy plant growth.

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Nitrate Sensor Design

E. coli native sensing system is the NarX/NarL two-component system

Sensing histadine kinase (NarL) is membrane bound, cross-phosphorylates itself in response to nitrate, then phorsphorylates a bound response regulator (NarX). There are probably good references for this, but idk them off the top of my head. Any paper involving this system will have this info though). The phosphorylated response regulator binds and represses a cognate promoter. In our case, this is promoter pNarL3 (which Nikola designed… I think he designed it such that the biding sites of NarX~P are in the -10 and -35 boxes of the promoter, so that they repress transcription, but I am not sure. I will ask him and get back to you).

These 2 proteins are in the E. coli chromosome, so we used a NarL knockout (we think sensitivity is dependent on NarL, not NarX)

We then put NarL under aTc inducible control on a ColE1 (high-copy_ plasmid)

We wanted to have an inverted sensor (i.e. signal is created at low levels of nitrate)

Thus, we put sfGFP downstream of the repressible promoter pNarL 3.

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Experiment performed

We characterized the nitrate sensor in M9 media with a range of nitrate concentrations and for various levels of aTc (inducing different levels of NarL). We detected fluorescence after around 6 hours of growth (mid-log phase) on a Cytek flow cytometer.

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Results

-Nonzero values of aTc all gave roughly similar sensitivities, implying that NarL is in saturation. This also implies that we can tune sensitivity with aTc, given that we find the narrow sensitive range of NarL. Nik and I will do this experiment once we have our new plasmid, and if it works, it’ll be super exciting.

-We sensed nitrate well, and at really really low levels. This is exciting. A graph is below…

More text from Kenny... Do you want to put the flow data since it's pretty?