Team:HKUST/Group3

From 2009.igem.org

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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensoring">Odorant Sensoring</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/AttranctantProduction">Attranctant Production</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li>
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<p>Construction of Attractant production and Reporter pathway</p>
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<p>Construction of Attractant Production and Reporter Pathway</p>
    
    
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   To functionally express the yeast ARO9 gene and EGFP gene, we have designed a reporter and an attractant production constructs. Based on the fact that yeast endogenous FUS1 promoter can sense the signal generated by the receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed such a pathway that puts ARO9 gene (with the original promoter deleted) and EGFP gene under the control of the FUS1 promoter. We have also have control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector. </p>
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   To functionally express the yeast ARO9 gene and EGFP gene, we have designed reporter and attractant production constructs. Since yeast endogenous FUS1 promoter can sense the downstream signal generated by the ligand-binding receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed a pathway that puts the ARO9 gene (with the original promoter deleted) and the EGFP gene under the control of the FUS1 promoter. We have also designed control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector. </p><br><br>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design1.jpg " width=180; height=80 /></a><br>
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&nbsp; (1) pRS426-FUS1P-ARO9-FUS1T, which is ARO9 gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p>
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<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure01.jpg" width=597; height=433 /><br>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design2.jpg " width=180; height=80 /></a><br>
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&nbsp; (2) pRS426-FUS1P-EGFP-FUS1T, which is EGFP gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/pRS426-EGFP.jpg" width=150; height=80 /></a><br>
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&nbsp; (3) pRS426-EGFP, which is EGFP gene without the FUS1 promoter, cloned into  the plasmid pRS406. </p>
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Figure 3. Attractant, reporter and control constructs</p>
Figure 3. Attractant, reporter and control constructs</p>
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<br><br>
<p>Test of the Reporter System </p>
<p>Test of the Reporter System </p>
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The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T, pRS426-EGFP, respectively. After α factor binding, we would expect to see that the MAPK pathway is activated and then the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy. </p>
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The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T and pRS426-EGFP respectively. After α factor binding, we would expect to see that the MAPK pathway is activated, leading to the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy. </p>
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<img src="https://static.igem.org/mediawiki/2009/c/c7/3Figure02.jpg" width=600; height=280 />
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/Table 1 Reporter functional assay and expected results.jpg " width=550; height=250 /></a><br>
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<p>Attractant Production Test</p>
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Table 1 Reporter functional assay and expected results. (a) is the YPH501 strain deleted with ARO9 gene and transformed with pRS426-FUS1P-EGFP-FUS1T; (b) is the same strain transformed with pRS426-EGFP. </p>
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We will test our attractant production pathway and the attractant effect on drosophila using the Cage Bioassays method given by J. Zhu, <em>et al</em>, 2003.(Figure 4) </p>
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<img src="https://static.igem.org/mediawiki/2009/5/52/3Figure03.jpg" width=600; height=370 />
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<p>Attractant production test</p>
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By comparing these six conditions, we can decide whether the attractant pathway under the FUS1 promoter can respond to signals generated by the receptor and effectively produce attractant molecules (Table 2). </p><br><br>
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Second we will test our attractant production pathway and the attractiveness towards Drosophilae using the Cage Bioassays  method given by J. Zhu, et al, 2003.(Figure 4) </p>
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<img src="https://static.igem.org/mediawiki/2009/9/90/3Figure04.jpg" width=600; height=300 /><br><br>
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<img src="http://igem2009hkust.fileave.com/wiki/Group3/test.jpg " width=400; height=230 /></a><br>
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<p>Further Characterization of the Attractant Production Pathway Function</p>
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   To further characterize the attractant production pathway function, we can do the following assays. <br><br>
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Figure 4, The cage bioassay to show the attractant effect. </p>
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   First, we can use the HPLC or other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards drosophila. <br><br>
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By comparing these six conditions, we can decide whether the attractant pathway under the FUS1 promoter can response to signals generated by the receptor and can effectively produce attractant molecules (table 2). </p>
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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Different traps and expected results.jpg " width=550; height=350 /></a><br>
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Table 2.  Different traps and expected results</p>
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<p>Further characterization of the Attractant production pathway function</p>
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   To further characterize the attractant production pathway function, we can do the following assays. <br>
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   First, we can use the HPLC or some other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards Drosophilae. <br>
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   Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production. </p>
   Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production. </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li>

Latest revision as of 03:44, 22 October 2009

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a

Construction of Attractant Production and Reporter Pathway

To functionally express the yeast ARO9 gene and EGFP gene, we have designed reporter and attractant production constructs. Since yeast endogenous FUS1 promoter can sense the downstream signal generated by the ligand-binding receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed a pathway that puts the ARO9 gene (with the original promoter deleted) and the EGFP gene under the control of the FUS1 promoter. We have also designed control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector.




Figure 3. Attractant, reporter and control constructs



Test of the Reporter System

The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T and pRS426-EGFP respectively. After α factor binding, we would expect to see that the MAPK pathway is activated, leading to the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy.




Attractant Production Test

We will test our attractant production pathway and the attractant effect on drosophila using the Cage Bioassays method given by J. Zhu, et al, 2003.(Figure 4)





By comparing these six conditions, we can decide whether the attractant pathway under the FUS1 promoter can respond to signals generated by the receptor and effectively produce attractant molecules (Table 2).





Further Characterization of the Attractant Production Pathway Function

To further characterize the attractant production pathway function, we can do the following assays.

First, we can use the HPLC or other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards drosophila.

Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production.




  • Background
  • Experimental Design
  • Parts Design
  • Experimental Results
  • Future Work
  • References
    • HKUST

    Retrieved from "http://2009.igem.org/Team:HKUST/Group3"