Difference between revisions of "Team:HKUST-Rice/Description"
Stephanieyiu (Talk | contribs) |
|||
| (5 intermediate revisions by 4 users not shown) | |||
| Line 33: | Line 33: | ||
<div id="bannerContainer"> | <div id="bannerContainer"> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/ | + | <img src="https://static.igem.org/mediawiki/2015/c/c6/Team_HKUST-Rice_2015_overviewslider.jpg" style="width: 100%; position: relative;"> |
<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> | <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> | ||
| Line 63: | Line 63: | ||
<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> | <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"> | <div class="project_image"> | ||
| − | <img style="width: | + | <img style="width:50%" src="https://static.igem.org/mediawiki/2015/f/f3/Team_HKUST-Rice_2015_pyearconstruct.PNG" alt="image caption"> |
</div> | </div> | ||
<div class="des"> | <div class="des"> | ||
| Line 71: | Line 71: | ||
<h1>Phosphate Sensor</h1> | <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> | <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: | + | <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"> | <div class="des"> | ||
| − | <p style="font-size:110%; float:left; padding-top:0px;"><strong>Figure 2. Phosphate sensor constructs.</strong> ( | + | <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> | <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Phosphate_Sensor"> Learn more ... </a></p> | ||
| Line 79: | Line 79: | ||
<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> | <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> | ||
<div class="project_image"> | <div class="project_image"> | ||
| − | <img style="width: | + | <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"> |
</div> | </div> | ||
<div class="des"> | <div class="des"> | ||
| Line 112: | Line 112: | ||
<div class="project_image"> | <div class="project_image"> | ||
| − | <img src="https://static.igem.org/mediawiki/2015/ | + | <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> | ||
<div class="des"> | <div class="des"> | ||
| − | <p style="font-size:110%"><strong>Figure 5. Working model of DIY Gel Imaging Station.</strong> Inlet shows the | + | <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> | <p style=" text-align: right"><a href="https://2015.igem.org/Team:HKUST-Rice/Design"> Learn more ... </a></p> | ||
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.
Figure 1. Construct for nitrate sensing. PyeaR with GFP generator.
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.
Figure 2. Phosphate sensor constructs. (left) PphoBR with GFP generator and (right) PphoA with GFP generator.
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.
Figure 1. K+ sensing construct with reporter.
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.
Figure 4. Brief diagrams of single and double constructs.
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.
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.
Figure 5. Working model of DIY Gel Imaging Station. Inlet shows the 1st gel image taken from the station
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.