Difference between revisions of "Team:HKUST-Rice/Nitrate Sensor PyeaR"
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| − | <p><i> Escherichia coli</i> (<i>E. coli</i> ) detects environmental nitrate by the <i>yeaR-yoaG</i> operon. According to Figure 1, P<sub>yeaR</sub> (Lin, <i>et al.</i>, 2007) is regulated by the Nar two-component regulatory system ( | + | <p><i> Escherichia coli</i> (<i>E. coli</i>) detects environmental nitrate by the <i>yeaR-yoaG</i> operon. According to Figure 1, P<sub>yeaR</sub> (Lin, <i>et al.</i>, 2007) is regulated by the Nar two-component regulatory system (Nohno <i>et al.</i>, 1989; Li et al., 1987) and NsrR regulatory protein (Partridge <i>et al.</i>, 2009). When there is nitrate or nitrite, the repression from the Nar system on P<sub>yeaR</sub> will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide by nitrate reductase. Nitric oxide will bind to the NsrR protein and relieve the repression on P<sub>yeaR</sub>. As a result, any genes that are downstream of P<sub>yeaR</sub> will be expressed. |
| − | <br><br>By ligating the promoter together with the GFP generator (<a href="http://parts.igem.org/Part:BBa_K381001"target="_blank">pSB1C3-BBa_K381001</a>), upward | + | <br><br>By ligating the promoter together with the GFP generator (<a href="http://parts.igem.org/Part:BBa_K381001"target="_blank">pSB1C3-BBa_K381001</a>), an upward trend for the reporter signal with increasing nitrate concentrations was expected.</p> |
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<h1>Experiment performed</h1> | <h1>Experiment performed</h1> | ||
<p>Two sets of characterization on <a href="http://parts.igem.org/Part:BBa_K381001"target="_blank"> pSB1C3-BBa_K381001</a> (<a href="https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR"target="_blank">BCCS-Bristol iGEM 2010</a>) in two different growth media, Luria Broth (LB) medium and M9 minimal medium were performed. M9 minimal medium was used as it does not contain nitrate and has a lower auto-fluorescence level, thus providing more accurate results. | <p>Two sets of characterization on <a href="http://parts.igem.org/Part:BBa_K381001"target="_blank"> pSB1C3-BBa_K381001</a> (<a href="https://2010.igem.org/Team:BCCS-Bristol/Wetlab/Part_Design/BioBricks/PyeaR"target="_blank">BCCS-Bristol iGEM 2010</a>) in two different growth media, Luria Broth (LB) medium and M9 minimal medium were performed. M9 minimal medium was used as it does not contain nitrate and has a lower auto-fluorescence level, thus providing more accurate results. | ||
| − | Potassium nitrate (KNO<sub>3</sub>) was used as a source of nitrate in the experiments. <i>E. coli</i> strain DH10B was used in the characterization of the promoter. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader. All experiments were conducted three times | + | Potassium nitrate (KNO<sub>3</sub>) was used as a source of nitrate in the experiments. <i>E. coli</i> strain DH10B was used in the characterization of the promoter. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader. All experiments were conducted three times on different days and the final results were obtained by combining the 3 characterization results together.</p> |
<p>Please visit <a href="https://static.igem.org/mediawiki/2015/6/6d/Team_HKUST-Rice_2015_2_YeaRp_Experiment_Protocol.pdf"target="_blank">P<sub>yeaR</sub> Experiment Protocol</a> for more details. </p> | <p>Please visit <a href="https://static.igem.org/mediawiki/2015/6/6d/Team_HKUST-Rice_2015_2_YeaRp_Experiment_Protocol.pdf"target="_blank">P<sub>yeaR</sub> Experiment Protocol</a> for more details. </p> | ||
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<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%"><strong>Figure 2. Characterization of P<sub>yeaR</sub> in LB.</strong> Bacteria hosting pSB1C3-BBa_K381001 | + | <p style="font-size:110%"><strong>Figure 2. Characterization of P<sub>yeaR</sub> in LB.</strong> Bacteria hosting pSB1C3-BBa_K381001 were grown overnight at 37<sup>o</sup>C in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspended in LB medium with different concentrations and grew for another 2.5 hours in a 37<sup>o</sup>C incubator with shaking. GFP emission measurements were made using an EnVision multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days.</p> </div> |
<p></p> | <p></p> | ||
<p> | <p> | ||
| − | According to Figure 2a, the relative fluorescence level increases | + | According to Figure 2a, the relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate. Furthermore, a plateau was shown from the 10 mM nitrate concentration point. The result obtained was as expected, according to previous experiments by the Edinburgh iGEM 2009 team and the BCCS-Bristol iGEM 2010 team, where the dynamic range of P<sub>yeaR</sub> was from 0-10 mM nitrate concentration. |
<br><br>After obtaining the results of P<sub>yeaR</sub> response behavior within 0-50 mM nitrate concentration, it shows that the fluorescence signal increases sharply between 0-10 mM nitrate concentration. Therefore, another characterization was done focusing on this concentration range.</p> | <br><br>After obtaining the results of P<sub>yeaR</sub> response behavior within 0-50 mM nitrate concentration, it shows that the fluorescence signal increases sharply between 0-10 mM nitrate concentration. Therefore, another characterization was done focusing on this concentration range.</p> | ||
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<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%"><strong>Figure 3. Characterization of P<sub>yeaR</sub> in M9.</strong> Bacteria hosting pSB1C3-BBa_K381001 | + | <p style="font-size:110%"><strong>Figure 3. Characterization of P<sub>yeaR</sub> in M9.</strong> Bacteria hosting pSB1C3-BBa_K381001 were grown overnight at 37<sup>o</sup>C in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspend in M9 medium with different concentrations and grew for another 4.5 hours in a 37<sup>o</sup>C incubator with shaking. GFP emission measurements were made using an EnVision multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days. |
</div> | </div> | ||
| − | <p>According to Figure 3a, the relative fluorescence level increases | + | <p>According to Figure 3a, the relative fluorescence level increases 4.37 folds from 0 μM and 1000 μM nitrate concentrations, and a plateau was shown from 500 μM nitrate concentration point. |
<br><br>After obtaining the results of P<sub>yeaR</sub> response behavior in the concentrations of 0-1000 μM nitrate, we find that the relative fluorescence level increases sharply between 0-500 μM concentrations of nitrate. As a result, another characterization was done focusing on this concentration range.</p> | <br><br>After obtaining the results of P<sub>yeaR</sub> response behavior in the concentrations of 0-1000 μM nitrate, we find that the relative fluorescence level increases sharply between 0-500 μM concentrations of nitrate. As a result, another characterization was done focusing on this concentration range.</p> | ||
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<p>Since we were concerned that endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed. | <p>Since we were concerned that endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed. | ||
| − | <br><br>With P<sub>araBAD</sub> as an inducible promoter, experiments were carried out to find the concentration of L-arabinose which could reduce endogenous noise most effectively, so that the promoter could be more accurate in sensing.</p> | + | <br><br>With P<sub>araBAD</sub> as an inducible promoter, experiments were carried out to find the concentration of L-arabinose which could reduce endogenous noise most effectively, so that the promoter could be more accurate in sensing nitrate concentrations.</p> |
<div class="project_image"> | <div class="project_image"> | ||
<img src="https://static.igem.org/mediawiki/2015/4/41/Team_HKUST-Rice_2015_Debug_mechanism.PNG" alt="image caption"> | <img src="https://static.igem.org/mediawiki/2015/4/41/Team_HKUST-Rice_2015_Debug_mechanism.PNG" alt="image caption"> | ||
</div> | </div> | ||
<p style="font-size:180%"><b>Rationale</b></p> | <p style="font-size:180%"><b>Rationale</b></p> | ||
| − | <p id="Methods">As P<sub>yeaR</sub> is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, | + | <p id="Methods">As P<sub>yeaR</sub> is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, endogenous nitrate alone will not be able to drive the transcription of P<sub>yeaR</sub>. On the other hand, when there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein, and transcription would result. With this method, less effects from the endogenous noise on the promoter is expected.</p> |
<p style="font-size:180%"><b>Result</b></p> | <p style="font-size:180%"><b>Result</b></p> | ||
<div class="project_image"> | <div class="project_image"> | ||
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<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%"><strong>Figure 5. Endogenous noise reduction of P<sub>yeaR</sub>.</strong> 0.01mM, 0.1mM, 1mM and 10mM of L-arabinose was added for inducing P<sub>araBAD</sub>. With different | + | <p style="font-size:110%"><strong>Figure 5. Endogenous noise reduction of P<sub>yeaR</sub>.</strong> 0.01mM, 0.1mM, 1mM and 10mM of L-arabinose was added for inducing P<sub>araBAD</sub>. With different concentrations of NsrR protein produced, the endogenous noise was reduced accordingly.</p></div> |
<p>According to Figure 5, with L-arabinose added, the curve shifts downwards. However, as the result obtained is similar to that of <a href="https://2015.igem.org/Team:HKUST-Rice/Expression#co-expression">co-expression</a>, it is uncertain that the downward shifting was due to co-expression of promoters or the method for endogenous noise reduction. </p> | <p>According to Figure 5, with L-arabinose added, the curve shifts downwards. However, as the result obtained is similar to that of <a href="https://2015.igem.org/Team:HKUST-Rice/Expression#co-expression">co-expression</a>, it is uncertain that the downward shifting was due to co-expression of promoters or the method for endogenous noise reduction. </p> | ||
</div> | </div> | ||
Revision as of 16:46, 9 September 2015
Nitrate Sensor - PyeaR
Nitrate as a Macro-nutrient
Nitrate is an essential nutrient which plays multiple roles in plant growth and reproduction. For example, it provides nitrogen that plants need for producing amino acids and nucleic acids (DNA and RNA). Also, it is a component of chlorophyll and is therefore essential for photosynthesis. Lack of nitrogen will lead to stunted growth, yellowing of leaves, etc.
Nitrate sensor Design
Escherichia coli (E. coli) detects environmental nitrate by the yeaR-yoaG operon. According to Figure 1, PyeaR (Lin, et al., 2007) is regulated by the Nar two-component regulatory system (Nohno et al., 1989; Li et al., 1987) and NsrR regulatory protein (Partridge et al., 2009). When there is nitrate or nitrite, the repression from the Nar system on PyeaR will be relieved due to the binding between the two. On the other hand, some nitrate will be converted into nitric oxide by nitrate reductase. Nitric oxide will bind to the NsrR protein and relieve the repression on PyeaR. As a result, any genes that are downstream of PyeaR will be expressed.
By ligating the promoter together with the GFP generator (pSB1C3-BBa_K381001), an upward trend for the reporter signal with increasing nitrate concentrations was expected.
Experiment performed
Two sets of characterization on pSB1C3-BBa_K381001 (BCCS-Bristol iGEM 2010) in two different growth media, Luria Broth (LB) medium and M9 minimal medium were performed. M9 minimal medium was used as it does not contain nitrate and has a lower auto-fluorescence level, thus providing more accurate results. Potassium nitrate (KNO3) was used as a source of nitrate in the experiments. E. coli strain DH10B was used in the characterization of the promoter. Quantitative characterization on the promoter was done by measuring the fluorescence signal intensity using an EnVision multilabel reader. All experiments were conducted three times on different days and the final results were obtained by combining the 3 characterization results together.
Please visit PyeaR Experiment Protocol for more details.
Results
After obtaining the quantitative results of GFP signal intensity using an EnVision multilabel reader, we processed the data with relative fluorescence level (in OD600) against nitrate concentration.
Dynamic range Characterization of PyeaR in LB
Figure 2. Characterization of PyeaR in LB. Bacteria hosting pSB1C3-BBa_K381001 were grown overnight at 37oC in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspended in LB medium with different concentrations and grew for another 2.5 hours in a 37oC incubator with shaking. GFP emission measurements were made using an EnVision multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days.
According to Figure 2a, the relative fluorescence level increases 7.21 folds between 0 mM and 10 mM concentrations of nitrate. Furthermore, a plateau was shown from the 10 mM nitrate concentration point. The result obtained was as expected, according to previous experiments by the Edinburgh iGEM 2009 team and the BCCS-Bristol iGEM 2010 team, where the dynamic range of PyeaR was from 0-10 mM nitrate concentration.
After obtaining the results of PyeaR response behavior within 0-50 mM nitrate concentration, it shows that the fluorescence signal increases sharply between 0-10 mM nitrate concentration. Therefore, another characterization was done focusing on this concentration range.
According to Figure 2b, the relative fluorescence level increases 4.23 folds between 0 mM and 10 mM nitrate concentrations. It shows an upward slope from 0 mM to 6 mM nitrate concentration, however, at 8 mM nitrate concentration point, it shows a downward slope. It then rises again at 10 mM nitrate. The result obtained is unexpected as according to previous experiments by the BCCS-Bristol iGEM 2010 team, where a continuous upward slope was obtained from 0 mM to 9 mM nitrate concentration. The discrepancy between the obtained and reference results could be due to the use of different bacterial strains. The strain used by the BCCS-Bristol iGEM 2010 team was MG1655, which may have been the cause in the promoter behavior difference.
Dynamic range Characterization of PyeaR in M9
Figure 3. Characterization of PyeaR in M9. Bacteria hosting pSB1C3-BBa_K381001 were grown overnight at 37oC in LB. They were then washed with 0.85% NaCl solution. Washed candidates were resuspend in M9 medium with different concentrations and grew for another 4.5 hours in a 37oC incubator with shaking. GFP emission measurements were made using an EnVision multilabel reader. This result was obtained by combining 3 charaterization data obtained in 3 different days.
According to Figure 3a, the relative fluorescence level increases 4.37 folds from 0 μM and 1000 μM nitrate concentrations, and a plateau was shown from 500 μM nitrate concentration point.
After obtaining the results of PyeaR response behavior in the concentrations of 0-1000 μM nitrate, we find that the relative fluorescence level increases sharply between 0-500 μM concentrations of nitrate. As a result, another characterization was done focusing on this concentration range.
According to Figure 3b, the relative fluorescence level increases 3.12 folds between 0 and 500 μM. Furthermore, with higher nitrate concentration, the relative fluorescence level increases accordingly. The result obtained is expected as according to the previous characterization with concentration gradient of 0 to 1000 μM nitrate, which also shows an upward slope between 0 to 500 μM.
Further Improvements
Since we were concerned that endogenous nitrate would affect the sensitivity of the promoter, a method in reducing the endogenous noise was designed.
With ParaBAD as an inducible promoter, experiments were carried out to find the concentration of L-arabinose which could reduce endogenous noise most effectively, so that the promoter could be more accurate in sensing nitrate concentrations.
Rationale
As PyeaR is regulated by the Nar system and NsrR protein, by overexpressing NsrR protein, endogenous nitrate alone will not be able to drive the transcription of PyeaR. On the other hand, when there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein, and transcription would result. With this method, less effects from the endogenous noise on the promoter is expected.
Result
Figure 5. Endogenous noise reduction of PyeaR. 0.01mM, 0.1mM, 1mM and 10mM of L-arabinose was added for inducing ParaBAD. With different concentrations of NsrR protein produced, the endogenous noise was reduced accordingly.
According to Figure 5, with L-arabinose added, the curve shifts downwards. However, as the result obtained is similar to that of co-expression, it is uncertain that the downward shifting was due to co-expression of promoters or the method for endogenous noise reduction.
References
Li, S. F., & DeMoss, J. A. (1987). Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.Journal of bacteriology, 169(10), 4614-4620.
Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG operon transcription by the nitrate-responsive regulator NarL is independent of oxygen-responsive regulator Fnr in Escherichia coli K-12. Journal of bacteriology, 189(21), 7539-7548.
Nohno, T., Noji, S., Taniguchi, S., & Saito, T. (1989). The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes. Nucleic acids research,17(8), 2947-2957.
Partridge, J. D., Bodenmiller, D. M., Humphrys, M. S., & Spiro, S. (2009). NsrR targets in the Escherichia coli genome: new insights into DNA sequence requirements for binding and a role for NsrR in the regulation of motility.Molecular microbiology, 73(4), 680-694.