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

 
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<div id= "page_title"><h1>Project</h1>
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<div id= "page_title" ><h1 id="scroll">Project</h1>
 
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<div class="project_row">
 
<h1>Overview</h1>
 
<h1>Overview</h1>
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<p>Nitrogen (N), phosphorus (P), and potassium (K) are three macronutrients for plants, and deficiencies in any of these can lead to plant diseases. By  
+
<p>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,
 
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  
+
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  
−
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  
+
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.</p>
 
of multiple outputs in a single system.</p>
 
 
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<div class="project_row">
 
<div class="project_row">
 
<hr class="para">
 
<hr class="para">
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                                                <h1>Nitrate Sensor</h1>
 +
<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>
 +
<div class="project_image">
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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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</div>
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<div class="des">
 +
<p style="font-size:110%"><strong>Figure 1. Construct for nitrate sensing. </strong> <i>P<sub>yeaR</sub></i> with GFP generator.</p></div>
 +
      <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"> Learn more ... </a></p>
 +
<br><br>
 +
<h1>Phosphate Sensor</h1>
 +
<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> 
 +
<img style="width:90%;" src="https://static.igem.org/mediawiki/2015/4/42/Team_HKUST-Rice_2015_phosphate_constructs.png" alt="image caption"><br><br>
 +
<div class="des">
 +
<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>
 +
        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Phosphate_Sensor"> Learn more ... </a></p>
 
 
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<table>
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<br><br><h1>Potassium Sensor</h1>
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<tr>
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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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<td style= "padding-left: 20px; ">
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<div class="project_image">
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<h1 style="line-height: 110%;">Potassium Sensor</h1>
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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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</td>
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−
+
−
<td style= "padding-left: 20px; ">
+
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<h1 style="line-height: 110%;">Phosphate Sensor</h1>
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    </td>
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−
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    <td style= "padding-left: 20px; "> 
+
−
<h1>Nitrate Sensor</h1>
+
−
</td>
+
−
</tr>
+
−
 
+
−
 
+
−
<tr>
+
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<td style= "padding-left: 20px; vertical-align: text-top;">
+
−
 
+
−
    <p><i>KdpFABC</i> transporter is a high affinity K<sup>+</sup> uptake system (Siebers, A. & Altendorf, K., 1988). The promoter upstream of <i>kdpFABC</i> operon,
+
−
<a href ="https://2015.igem.org/Team:HKUST-Rice/Potassium_Sensor" ><i>kdpFp</i></a>, works under low K<sup>+</sup> concentrations (Polarek, J. W., et al., 1992; Walderhaug, M. O., et al., 1992). The goal is to characterize  
+
−
<i>kdpFp</i> and build a device which is able to sense different concentrations of K<sup>+</sup> and express different levels of GFP accordingly.<br><br>
+
−
+
−
</td>
+
−
+
−
<td style= "padding-left: 20px; vertical-align: text-top;">
+
−
 
+
−
<p><a href ="https://2015.igem.org/Team:HKUST-Rice/Phosphate_Sensor" ><i>phoAp</i> and <i>phoBRp</i></a> promoters (Hsieh, Y. J., & Wanner, B. L., 2010) are cross-regulated by <i>phoB</i> and <i>phoR</i>, and are
+
−
usually repressed under high phosphate concentrations. <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 activate the
+
−
            <i>phoAp</i> and <i>phoBRp</i> promoters. <br><br>
+
−
    </td>
+
−
+
−
    <td style= "padding-left: 20px; vertical-align: text-top;"> 
+
−
<p><a href ="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor_PyeaR"><i>yeaRp</i></a> promoter (Lin, et al., 2007) is normally cross-regulated by the Nar two-component regulatory system (T.Nohno,
+
−
et al. , 1989) and <i>NsrR</i>, a regulatory protein. When there is nitrate and nitrite, it will be converted into nitric oxide.
+
−
The nitric oxide will bind to <i>NsrR</i> and relieve the repression on the <i>yeaRp</i> promoter. As a result, any
+
−
genes that are downstream of the <i>yeaRp</i> promoter will be expressed.</font><br><br>
+
−
</td>
+
−
</tr>
+
−
 
+
−
 
+
−
<tr>
+
−
    <td>
+
−
<img src= "https://static.igem.org/mediawiki/2015/b/b4/Team-HKUST-Rice-KdpdF_promoter_1.JPG" style="width: 100%; height: auto; float: center;"></p>
+
−
        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Potassium_Sensor"> Learn more ... </a></p>
+
−
</td>
+
−
+
−
<td>
+
−
    <img src= "https://static.igem.org/mediawiki/2015/7/7f/Team-HKUST-Rice-Pphoa_characterization.JPG" style="width: 100%; height: auto; float: center;"></p>
+
−
        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Phosphate_Sensor"> Learn more ... </a></p>
+
−
    </td>
+
−
+
−
    <td> 
+
−
      <img src= "https://static.igem.org/mediawiki/2015/0/09/Team-HKUST-Rice-Pyear_characterization.JPG" style="width: 100%; height: auto; float: center;"></p>
+
−
      <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Nitrate_Sensor"> Learn more ... </a></p>
+
−
</td>
+
−
</tr>
+
−
 
+
−
</table>
+
 
</div>
 
</div>
 +
                                        <div class="des">
 +
<p style="font-size:110%; padding-left:6%;"><strong>Figure 1. K<sup>+</sup> sensing construct with reporter.</strong></p></div>
 +
        <p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Potassium_Sensor"> Learn more ... </a></p></div>
 
 
 +
 
<div class="project_row">
 
<div class="project_row">
−
<hr class="para">
+
<hr class="para">
−
<h1>Signal Co-expression</h1>
+
<h1>Parallel Sensors</h1>
−
<p>In order to characterize the output difference between a single expression system and co-expression  
+
<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>
−
from one vector, we will be constructing several inducible systems that give off fluorescence output and compare the dose  
+
<br><br>
−
response of the individual systems in the two scenarios.<br><br>As part of the expression platform, we also aim to construct a
+
  <div class="project_image">
−
3-input AND logic gate using toehold switches (Green, A. A., et al., 2014) to integrate the input signals detected from the three
+
<img src="https://static.igem.org/mediawiki/2015/c/ce/Team_HKUST-Rice_2015_Coex111.PNG" alt="image caption">
−
(N, P and K) promoters.</font><br>
+
−
  <img src= "https://static.igem.org/mediawiki/2015/0/08/Team-HKUST-Rice-3inputlogic_gate.JPG" style="width: 400px; height: auto; float: center;"></p>
+
−
  <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Expression"> Learn more ... </a></p>
+
−
+
 
</div>
 
</div>
 +
 +
<div class="des">
 +
<p style="font-size:110%"><strong>Figure 4. Brief diagrams of single and double constructs.</strong></p></div>
 
 
−
+
 
−
<div class="project_row">
+
  <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Expression"> Learn more ... </a></p>
−
                                        <hr class="para">
+
<h1><i>P<sub>araBAD<sub></i></h1>
−
<h1>References:</h1>
+
 
<p>
 
<p>
−
<li class="ref">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>
+
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.
 +
<p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Expression/ParaBAD"> Learn more ... </a></p>
 +
</p>
 +
</div>
 +
<div class="project_row">
 +
<hr class="para">
 +
<h1>DIY Gel Imaging Station</h1>
 +
<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>
 +
 
 +
<div class="project_image">
 +
<img width=800px src="https://static.igem.org/mediawiki/2015/9/97/HKUST_Rice15_still_image_diy_gel_doc_webWithinlet.png" alt="image caption">
 +
</div>
 +
<div class="des">
 +
<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>
 +
 
 +
<p  style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Design"> Learn more ... </a></p>
 +
</div>
 +
</div>
 +
<div class="project_row">
 +
<hr class="para">
 +
<h2>References</h2>
 +
                                <p style="font-size:125%">
 +
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>
 
   
 
   
−
<li class="ref">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>
+
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>
  
−
<li class="ref">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>
+
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>
  
−
<li class="ref">Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.</li><br>
+
Siebers, A. and Altendorf, K. (1988). The K+-translocating Kdp-ATPase from Escherichia coli. European Journal of Biochemistry, 178, 131–140.<br><br>
  
−
<li class="ref">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.</li><br>
+
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>
 
   
 
   
−
<li class="ref">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.</li>
+
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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{{HKUST-Rice Directory}}
  
 
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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.