Difference between revisions of "Team:HKUST-Rice/Description"

 
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<p style="font-size:40px;color:#FFFFFF;line-height:1em;"> Potassium, Nitrate and Phosphate Biosensor </font><br>
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<a href=" #scroll"><img class="button" src="https://static.igem.org/mediawiki/2015/c/ce/HKUST-Rice15_bannerarrow.jpg" style="width: 4%; height: auto; margin-top: -6%; position: absolute; z-index: 999; left:48%; "></a>
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                                <font size= "5"> HKUST-Rice iGEM Team </font></p>
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<div id= "page_title" ><h1 id="scroll">Project</h1>
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<h1>Overview</h1>
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<p>Nitrogen (N), phosphorus (P) and potassium (K) are three plant macronutrients, and deficiencies in any of these can lead to plant diseases. By
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creating a biological sensor that can quickly provide soil status to plant owners, we can prevent plant diseases due to the lack of nutrients. In view of this,
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our team constructed a biological sensor in <i>E. coli</i>, which can detect NPK levels in the surrounding environment and give responses in the form of
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colors. In addition, we characterized the effects of a dual output system, in contrast to a single output system, in order to anticipate the expression
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of multiple outputs in a single system.</p>
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                                                <h1>Nitrate Sensor</h1>
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<p><i>P<sub>yeaR</sub></i> is normally cross-regulated by the Nar two-component regulatory system (Nohno et al., 1989; Lin et al., 2007) and NsrR, a regulatory protein. When there is nitrate and nitrite, it will be converted into nitric oxide. The nitric oxide will bind to NsrR and relieve the repression on the <i>P<sub>yeaR</sub></i>. As a result, any genes that are downstream of the <i>P<sub>yeaR</sub></i> promoter will be expressed.</p>
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<img style="width:50%" src="https://static.igem.org/mediawiki/2015/f/f3/Team_HKUST-Rice_2015_pyearconstruct.PNG" alt="image caption">
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<p style="font-size:110%"><strong>Figure 1. Construct for nitrate sensing. </strong> <i>P<sub>yeaR</sub></i> with GFP generator.</p></div>
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      <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"> Learn more ... </a></p>
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<br><br>
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<h1>Phosphate Sensor</h1>
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<p><i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i></a> promoters  are cross-regulated by <i>phoB</i> and <i>phoR</i> and repressed under high phosphate concentrations (Hsieh & Wanner, 2010). PhoR behaves as an activator as well as an inactivator for PhoB. When phosphate is limited, PhoR will phosphorylate PhoB and the phosphorylated PhoB will directly activate the <i>P<sub>phoA</sub></i> and <i>P<sub>phoBR</sub></i> promoters. </p> 
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<p style="font-size:110%; float:left; padding-top:0px;"><strong>Figure 2. Phosphate sensor constructs.</strong> (left) <i>P<sub>phoBR</sub></i> with GFP generator and (right) <i>P<sub>phoA</sub></i> with GFP generator.</strong></p></div>
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        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Phosphate_Sensor"> Learn more ... </a></p>
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<br><br><h1>Potassium Sensor</h1>
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<p><p><i>kdpFABC</i> transporter is a high affinity K<sup>+</sup> uptake system (Siebers & Altendorf, 1988). The promoter upstream of <i>kdpFABC</i> operon,<i>P<sub>kdpF</sub></i>, works under low K<sup>+</sup> concentrations (Polarek et al., 1992; Walderhaug et al., 1992). The goal is to characterize <i>P<sub>kdpF</sub></i> and build a device which is able to sense different concentrations of K<sup>+</sup> and express different levels of GFP accordingly.<p>
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<img style="width: 50%; height: auto; float: center;" src="https://static.igem.org/mediawiki/2015/4/42/Team_HKUST-Rice_2015_potassium_figure_1.png" alt="image caption">
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                                        <div class="des">
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<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. K<sup>+</sup> sensing construct with reporter.</strong></p></div>
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        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Potassium_Sensor"> Learn more ... </a></p></div>
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<h1>Parallel Sensors</h1>
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<p>In order to characterize the output difference between a single expression system and co-expression system from one vector, we built two inducible systems and compared the dose response of each of the individual system in a single construct plasmid to that of a double constructs plasmid. Fluorescence was used as the readout.  </font><br></p>
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<p style="font-size:110%"><strong>Figure 4. Brief diagrams of single and double constructs.</strong></p></div>
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  <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Expression"> Learn more ... </a></p>
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<h1><i>P<sub>araBAD<sub></i></h1>
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In the parallel sensors we build one of the sensor using <i>P<sub>araBAD<sub></i>. However we discover that there are different opinions on the mode of response of this widely used promoter. As such we decided to investigate on this promoter.
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<p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Expression/ParaBAD"> Learn more ... </a></p>
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<h1>DIY Gel Imaging Station</h1>
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<p>Gel electrophoresis is an important tool for biologist, an imaging station that is easily accessible is crucial for a productivity of any iGEM team. In view of this, our team made a DIY Gel Imaging Station using cheap and easy-to-obtain materials.</p>
  
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<p><font size= "6" color=#000000>Overview</font></p><br>
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<p><font size= "4" color=#000000> Nitrogen (N), Phosphorus (P), and potassium (K) are three macronutrients for plants, and deficiencies in any of these can lead to plant diseases. By creating a biological sensor that can quickly provide soil status to plant owners, we can prevent plant diseases due to the lack of nutrients. In view of this, our team is constructing a biological sensor in <i>E. coli</i>, which can detect NPK levels in the surrounding environment and give responses in the form of colors. In addition, we are characterizing the effects of a dual output system, in contrast to a single output system, in order to anticipate the expression of multiple outputs in a single system.</font></p><br>
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<img width=800px src="https://static.igem.org/mediawiki/2015/9/97/HKUST_Rice15_still_image_diy_gel_doc_webWithinlet.png" alt="image caption">
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<p style="font-size:110%"><strong>Figure 5. Working model of DIY Gel Imaging Station.</strong> Inlet shows the 1<sup>st</sup> gel image taken from the station</p></div>
  
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<p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Design"> Learn more ... </a></p>
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    <td><br><img src= "https://static.igem.org/mediawiki/2015/b/b4/Team-HKUST-Rice-KdpdF_promoter_1.JPG"></td>
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    <td><br><br><br><p><font size= "3" color=#000000>KdpFABC transporter is a high affinity K+ uptake system (Siebers, A. & Altendorf, K., 1988). The promoter upstream of kdpFABC operon, kdpFp, works under low K+ concentration (Polarek, J. W., et al., 1992; Walderhaug, M. O., et al., 1992). The goal is to characterize kdpFp and build a device which is able to sense different concentration of K+ and express different level of GFP accordingly.</p></td>
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<h2>References</h2>
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    <td><br><p><font size= "3" color=#000000>For phosphate sensing, we decided to use <i>P<sub>phoA</sub></i> promoter (Hsieh, Y. J., & Wanner, B. L.,2010).. <i>P<sub>phoA</sub></i> promoter is cross-regulated by <i>phoB</i>, a DNA binding response regulator and <i>phoR</i>, a sensory histidine kinase, and is usually repressed under high phosphate concentration. <i>phoR</i> behaves as an activator as well as an inactivator for <i>phoB</i>. When phosphate is limited, <i>phoR</i> will phosphorylate <i>phoB</i> and the phosphorylated <i>phoB</i> will directly activates the expression of <i>P<sub>phoA</sub></i> promoter. In contrast, when in high concentration of phosphate, <i>phoR</i> will dephosphorylate <i>phoB</i> and thus, inactivates the expression of <i>P<sub>phoA</sub></i> promoter. As the biobrick of <i>P<sub>phoA</sub></i> promoter with a GFP coding device submitted is not released, we have to clone the promoter from <i>E. coli</i> strain DH10B.</font></p></td>  
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  <td><br><img src= "https://static.igem.org/mediawiki/2015/7/7f/Team-HKUST-Rice-Pphoa_characterization.JPG"></td>
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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.<br><br>
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  <td><br><p><font size= "3" color=#000000>For nitrate sensing, we decided to use <i>P<sub>yeaR</sub></i> promoter (Lin, et al, 2007). It is normally cross-regulated by the Nar two-component regulatory system (T.Nohno, et,al. , 1989) and <i>nsrR</i>, a regulatory protein that prevents the transcription of a number of operons in <i>E. coli</i>. When there is nitrate and nitrite in the environment, it will enter the cell and then being converted into nitric oxide. The nitric oxide will bind to <i>nsrR</i> and relieve the repression on the <i>P<sub>yeaR</sub></i> promoter. As a result, any genes that are downstream of the <i>P<sub>yeaR</sub></i> promoter will be expressed. As the biobrick of <i>P<sub>yeaR</sub></i> promoter with a GFP coding device has already been submitted by the University of Bristol in 2010, we could characterize it and directly apply to our biosensor.</font></p><br></td>
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<p><font size= "3" color=#000000>Reference:
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<li>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.</li><br>
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<li>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.</li><br>
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Lin, H. Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of <i>yeaR-yoaG</i> 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.<br><br>
  
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<li>Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.</li><br>
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Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.<br><br>
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<li>Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.</li></p>
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Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.<br><br>
  
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Walderhaug, M. O., Polarek, J. W., Voelkner, P., Daniel, J. M., Hesse, J. E., Altendorf, K., & Epstein, W. (1992). KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. Journal of Bacteriology, 174(7), 2152–2159.<br><br>
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Green, A.A., Silver, P.A., Collins, J.J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 157(4), 925-935.
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Latest revision as of 02:43, 19 September 2015

Project

Overview

Nitrogen (N), phosphorus (P) and potassium (K) are three plant macronutrients, and deficiencies in any of these can lead to plant diseases. By creating a biological sensor that can quickly provide soil status to plant owners, we can prevent plant diseases due to the lack of nutrients. In view of this, our team constructed a biological sensor in E. coli, which can detect NPK levels in the surrounding environment and give responses in the form of colors. In addition, we characterized the effects of a dual output system, in contrast to a single output system, in order to anticipate the expression of multiple outputs in a single system.

Nitrate Sensor

PyeaR is normally cross-regulated by the Nar two-component regulatory system (Nohno et al., 1989; Lin et al., 2007) and NsrR, a regulatory protein. When there is nitrate and nitrite, it will be converted into nitric oxide. The nitric oxide will bind to NsrR and relieve the repression on the PyeaR. As a result, any genes that are downstream of the PyeaR promoter will be expressed.

image caption

Figure 1. Construct for nitrate sensing. PyeaR with GFP generator.

Learn more ...



Phosphate Sensor

PphoA and PphoBR promoters are cross-regulated by phoB and phoR and repressed under high phosphate concentrations (Hsieh & Wanner, 2010). PhoR behaves as an activator as well as an inactivator for PhoB. When phosphate is limited, PhoR will phosphorylate PhoB and the phosphorylated PhoB will directly activate the PphoA and PphoBR promoters.

image caption

Figure 2. Phosphate sensor constructs. (left) PphoBR with GFP generator and (right) PphoA with GFP generator.

Learn more ...



Potassium Sensor

kdpFABC transporter is a high affinity K+ uptake system (Siebers & Altendorf, 1988). The promoter upstream of kdpFABC operon,PkdpF, works under low K+ concentrations (Polarek et al., 1992; Walderhaug et al., 1992). The goal is to characterize PkdpF and build a device which is able to sense different concentrations of K+ and express different levels of GFP accordingly.

image caption

Figure 1. K+ sensing construct with reporter.

Learn more ...

Parallel Sensors

In order to characterize the output difference between a single expression system and co-expression system from one vector, we built two inducible systems and compared the dose response of each of the individual system in a single construct plasmid to that of a double constructs plasmid. Fluorescence was used as the readout.



image caption

Figure 4. Brief diagrams of single and double constructs.

Learn more ...

ParaBAD

In the parallel sensors we build one of the sensor using ParaBAD. However we discover that there are different opinions on the mode of response of this widely used promoter. As such we decided to investigate on this promoter.

Learn more ...

DIY Gel Imaging Station

Gel electrophoresis is an important tool for biologist, an imaging station that is easily accessible is crucial for a productivity of any iGEM team. In view of this, our team made a DIY Gel Imaging Station using cheap and easy-to-obtain materials.

image caption

Figure 5. Working model of DIY Gel Imaging Station. Inlet shows the 1st gel image taken from the station

Learn more ...

References

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.

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.

Hsieh, Y. J., & Wanner, B. L. (2010). Global regulation by the seven-component P i signaling system. Current opinion in microbiology, 13(2), 198-203.

Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.

Walderhaug, M. O., Polarek, J. W., Voelkner, P., Daniel, J. M., Hesse, J. E., Altendorf, K., & Epstein, W. (1992). KdpD and KdpE, proteins that control expression of the kdpABC operon, are members of the two-component sensor-effector class of regulators. Journal of Bacteriology, 174(7), 2152–2159.

Green, A.A., Silver, P.A., Collins, J.J., & Yin, P. (2014). Toehold Switches: De-Novo-Designed Regulators of Gene Expression. Cell, 157(4), 925-935.