Difference between revisions of "Team:HKUST-Rice/Nitrate Sensor PdcuS"

 
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<div id= "page_title"><h1>Nitrate Sensor - <i>PdcuS</i></h1>
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<div id= "page_title"><h1>Nitrate Sensor - <i>P<sub>dcuS</sub></i></h1>
 
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                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
 
                       <a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"><img src="https://static.igem.org/mediawiki/2015/7/7a/HKUST-Rice15_rightarrow.png">
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>yeaRp</i>) </p></a>
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<p style="color:#5570b0; font-size: 130%"> Nitrate sensor (<i>P<sub>yeaR</sub></i>) </p></a>
 
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<h1>Nitrate as a Macro-nutrient</h1>
 
<h1>Nitrate as a Macro-nutrient</h1>
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<p>1. Nitrate is an essential macronutrient in plants <br>
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<p>Nitrate is an important nutrient for plant growth, as it allows them to make amino acids and in turn, proteins. To ensure good crop yield, farmers must ensure that their soil has sufficient nitrates. They often do this by adding (often too much) fertilizer to their soils.  
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2. Probably Wikipedia has some nice stuff about how it affects plant growth/healthy development, etc. <br>
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<br><br>Here we characterize a native <i>E. coli</i> nitrate sensor that can be used as a biological sensor on the field to detect soil nitrate levels, and thus prevent the overfertilization of fields and the negative environmental effects associated with that practice. <br></p>
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3. It is important that soil has nitrate to ensure healthy plant growth. <br></p>
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<h1>Nitrate Sensor Design</h1>
 
<h1>Nitrate Sensor Design</h1>
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<p>E. coli native sensing system is the NarX/NarL two-component system</p>
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<p>In order to develop and characterize a nitrate sensing system for <i>E. coli</i>, we utilized the native NarX-NarL two-component system. NarX is a sensory histadine kinase that selectively phosphorylates NarL, the cognate response regulator, in the presence of extracellular NO<sub>3</sub><sup>-</sup>. Phosphorylated NarL, in turn can bind to promoters to regulate downstream gene expression.</p>
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<p>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).</p>
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<p>These 2 proteins are in the E. coli chromosome, so we used a NarL knockout (we think sensitivity is dependent on NarL, not NarX)</p>
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<p>We then put NarL under aTc inducible control on a ColE1 (high-copy_ plasmid)</p>
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<p>We wanted to have an inverted sensor (i.e. signal is created at low levels of nitrate)</p>
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<p>Thus, we put sfGFP downstream of the repressible promoter pNarL 3.</p>
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<p><br><br>Although many promoters have been characterized and the consensus DNA-binding sequence is identified, most promoters also contain FNR sites and are regulated by other systems (Darwin & Tyson, 1997). We identified the promoter region upstream of DcuS as a potential promoter that recognizes NarL and is independent of other systems (Goh & Bledsoe et al., 2005). Based on the protein-DNA interactions described in Goh, we selected a 73bp region directly upstream of the +1 promoter site as a potential repressible promoter in the presence of NO<sub>3</sub><sup>-</sup>
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<br><br>pND16 was constructed based on pRS334 (courtesy of Tabor Lab, Rice University), a high-copy (ColE1 origin) plasmid in which expression of sfGFP is controlled by the <i>dcuS</i> promoter (P<sub>dcuS</sub>) and expression of NarL can be controlled via an aTc-inducible TetR-P<sub>tet</sub> one-component system. This allows us to regulate levels of cellular NarL compared to native NarL and NarX to tune the sensitivity and dynamic range of the sensor. Since NarL is chromosomally expressed in <i>E. coli</i>, this plasmid was transformed into a NarL knockout strain of BW29655.
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<h1>Experiment that we did</h1>
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<h1>Experiments and Results</h1>
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<p>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.</p>
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<p><strong>Finding the proper aTc induction level to activate the sensor</strong></p>
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<p>We wished to see at what levels of aTc the sensor performed at. Thus, we characterized the sensor’s fluorescent output for 5 different aTc concentrations. The data below indicates that at even our lowest nonzero aTc level, the system was saturated with NarL and thus was performing at its highest sensitivity.
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<br><br>We also noticed that there was a marked difference in fold change and in the K1/2 parameter for the system between 0 and 20 ng/mL aTc. This indicates that we may have built a sensor with tunable sensitivity, and was the inspiration for experiment 3.</p>
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                                  <p><strong>In depth-characterization of sensor</strong></p>
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                                  <p>At this point, we chose our lowest saturating aTc value (20 ng/mL) and created a 12-point NO<sub>3</sub><sup>-</sup> - fluorescence transfer function in order to better determine the system parameters.
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<br><br>A sigmoidal fit on the data below suggests that our system’s half-maximum occurs at .02 mM, equivalent to 1.25 ppm NO<sub>3</sub><sup>-</sup> in the soil. The system also has a fold change of 7.15.</p>
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                                <p><strong>Tuning Sensitivity and Fold-Change</strong></p>
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                                <p>Taking information from experiments 1 and 2, we decided to try changing the sensitivity and fold change of the system by picking aTc values between zero and 20 ng/mL, the previous saturating value.
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<br><br>We picked 6 different aTc values and 6 different nitrate levels for this characterization and arrived at the results below</p>
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<h1>Result obtained</h1>
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<h1>Conclusion</h1>
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<p>-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.
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<p>Our sensor works! It is sensitive enough to detect levels of nitrate below the typical levels in soil. By simply adding a chromophore as the reporter protein, thus system could be converted into a colorimetric nitrate sensor.  
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-We sensed nitrate well, and at really really low levels. This is exciting. A graph is below…
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<p>More text from Kenny... Do you want to put the flow data since it's pretty?</p>
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<h2>References</h2>
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  <p style="font-size:125%">Darwin, A., & Tyson, K. (1997). Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K12 depends on DNA binding site arrangement. Molecular …, 25, 583–595. Retrieved from http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1997.4971855.x/full
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<br><br>Goh, E. B., Bledsoe, P. J., Chen, L. L., Gyaneshwar, P., Stewart, V., & Igo, M. M. (2005). Hierarchical control of anaerobic gene expression in Escherichia coli K-12: The nitrate-responsive NarX-NarL regulatory system represses synthesis of the fumarate-responsive DcuS-DcuR regulatory system. Journal of Bacteriology, 187(14), 4890–4899.</p></div>
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{{HKUST-Rice Directory}}

Latest revision as of 15:14, 18 September 2015


Nitrate Sensor - PdcuS

Nitrate as a Macro-nutrient

Nitrate is an important nutrient for plant growth, as it allows them to make amino acids and in turn, proteins. To ensure good crop yield, farmers must ensure that their soil has sufficient nitrates. They often do this by adding (often too much) fertilizer to their soils.

Here we characterize a native E. coli nitrate sensor that can be used as a biological sensor on the field to detect soil nitrate levels, and thus prevent the overfertilization of fields and the negative environmental effects associated with that practice.

Nitrate Sensor Design

In order to develop and characterize a nitrate sensing system for E. coli, we utilized the native NarX-NarL two-component system. NarX is a sensory histadine kinase that selectively phosphorylates NarL, the cognate response regulator, in the presence of extracellular NO3-. Phosphorylated NarL, in turn can bind to promoters to regulate downstream gene expression.

image caption



Although many promoters have been characterized and the consensus DNA-binding sequence is identified, most promoters also contain FNR sites and are regulated by other systems (Darwin & Tyson, 1997). We identified the promoter region upstream of DcuS as a potential promoter that recognizes NarL and is independent of other systems (Goh & Bledsoe et al., 2005). Based on the protein-DNA interactions described in Goh, we selected a 73bp region directly upstream of the +1 promoter site as a potential repressible promoter in the presence of NO3-

pND16 was constructed based on pRS334 (courtesy of Tabor Lab, Rice University), a high-copy (ColE1 origin) plasmid in which expression of sfGFP is controlled by the dcuS promoter (PdcuS) and expression of NarL can be controlled via an aTc-inducible TetR-Ptet one-component system. This allows us to regulate levels of cellular NarL compared to native NarL and NarX to tune the sensitivity and dynamic range of the sensor. Since NarL is chromosomally expressed in E. coli, this plasmid was transformed into a NarL knockout strain of BW29655.

image caption

Experiments and Results

Finding the proper aTc induction level to activate the sensor

We wished to see at what levels of aTc the sensor performed at. Thus, we characterized the sensor’s fluorescent output for 5 different aTc concentrations. The data below indicates that at even our lowest nonzero aTc level, the system was saturated with NarL and thus was performing at its highest sensitivity.

We also noticed that there was a marked difference in fold change and in the K1/2 parameter for the system between 0 and 20 ng/mL aTc. This indicates that we may have built a sensor with tunable sensitivity, and was the inspiration for experiment 3.

image caption

In depth-characterization of sensor

At this point, we chose our lowest saturating aTc value (20 ng/mL) and created a 12-point NO3- - fluorescence transfer function in order to better determine the system parameters.

A sigmoidal fit on the data below suggests that our system’s half-maximum occurs at .02 mM, equivalent to 1.25 ppm NO3- in the soil. The system also has a fold change of 7.15.

image caption

Tuning Sensitivity and Fold-Change

Taking information from experiments 1 and 2, we decided to try changing the sensitivity and fold change of the system by picking aTc values between zero and 20 ng/mL, the previous saturating value.

We picked 6 different aTc values and 6 different nitrate levels for this characterization and arrived at the results below

image caption

Conclusion

Our sensor works! It is sensitive enough to detect levels of nitrate below the typical levels in soil. By simply adding a chromophore as the reporter protein, thus system could be converted into a colorimetric nitrate sensor.




References

Darwin, A., & Tyson, K. (1997). Differential regulation by the homologous response regulators NarL and NarP of Escherichia coli K12 depends on DNA binding site arrangement. Molecular …, 25, 583–595. Retrieved from http://onlinelibrary.wiley.com/doi/10.1046/j.1365-2958.1997.4971855.x/full

Goh, E. B., Bledsoe, P. J., Chen, L. L., Gyaneshwar, P., Stewart, V., & Igo, M. M. (2005). Hierarchical control of anaerobic gene expression in Escherichia coli K-12: The nitrate-responsive NarX-NarL regulatory system represses synthesis of the fumarate-responsive DcuS-DcuR regulatory system. Journal of Bacteriology, 187(14), 4890–4899.