Difference between revisions of "Team:HKUST-Rice/Nitrate Sensor PdcuS"
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| − | <h1>Nitrate | + | <h1>Nitrate Sensor Design</h1> |
| − | <p> | + | <p>E. coli native sensing system is the NarX/NarL two-component system<br> |
| − | + | 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).<br> | |
| − | + | These 2 proteins are in the E. coli chromosome, so we used a NarL knockout (we think sensitivity is dependent on NarL, not NarX)<br> | |
| − | + | We then put NarL under aTc inducible control on a ColE1 (high-copy_ plasmid)<br> | |
| + | We wanted to have an inverted sensor (i.e. signal is created at low levels of nitrate)<br> | ||
| + | Thus, we put sfGFP downstream of the repressible promoter pNarL 3.<br> | ||
| + | </p> | ||
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<img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption"> | <img src="https://static.igem.org/mediawiki/2015/b/be/HKUST-Rice15_Resultsbutton.png" alt="image caption"> | ||
Revision as of 19:59, 9 September 2015
Nitrate Sensor - PdcuS
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.
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.
Experiment that we did
Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.
Result obtained
Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.
Lorem ipsum dolor sit amet, pro aeque temporibus eu, eum qualisque assueverit te. Ad est admodum epicuri suscipit, te alterum aliquando adversarium usu, pro ex omnesque luptatum comprehensam. In vix alia percipit gloriatur, no ferri lorem aliquando cum. Fugit concludaturque sed ne, ea sumo dico adolescens eos, quo eu pertinax expetendis. An his omnes instructior, vide possim eam id. Te cum enim sale offendit, vocent copiosae luptatum ut per. Eam in alienum accusamus, et probo reque vix. Vivendum necessitatibus qui ad, no vis enim veniam perpetua. Eu pri habemus senserit, dicit tation expetenda usu et. Sea eu dolor deserunt dissentias, sed an oportere moderatius assueverit. Usu te tation gloriatur, vidit tollit utinam mea id.