Team:HKUST/Group3

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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li>
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project description</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Background">Background</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Experiment">Experimental design</a></li>
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<b>
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<span style="color:green">
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<li>Main Parts</li>
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</b>
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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/AttractantProduction">Attractant Production</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li>
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<span style="color:green">
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<li>Resources</li>
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</span>
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</b>
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Result">Experimental result</a></li>
 
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li>
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol list</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other resources</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Future">Future plan</a></li>
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<ul>
<ul>
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li>
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li>
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<li><a href="https://2009.igem.org/Team:Consolidation">Consolidation</a></li>
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<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li>
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li>
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li>
</ul>
</ul>
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<div class="contentatt_d"> <h3>a</h3>
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<h3>Welcome</h3>
 
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<p>Attractant production and reporter Construct</p>
 
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<p>The attractant production pathway contains three parts, which are FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from yeast genome and then cloned into PRS426 yeast expression vector for further functional assay. </p>
 
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<p>The reporter construct also contains three parts, which are FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into PRS426 yeast expression vector for further functional assay.</p>
 
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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design1.jpg " width=550; height=350 /></a>
 
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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design2.jpg " width=550; height=350 /></a>
 
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<p>I. Primer Design</p>
 
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<p>We have designed several sets of primers for our construction, which are listed in Table 1. These mainly consist of primers for FUS1 promoter amplification, EGFP reporter amplification, ARO9 gene amplification and FUS1 terminator amplification. Primer statistics are calculated using NetPrimer.</p>
 
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<p>Table 1  Primer sequences designed for the constructions</p>
 
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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 tablePrimer.jpg " width=550; height=700 /></a>
 
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<p>*Note:</p>
 
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<p> Restriction sites are highlighted in yellow.</p>
 
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<p>II. PCR</p>
 
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<p>Three out of four genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified out from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from plasmid YTK-6/Tpi/EGFP.</p>
 
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<p>III. Cloning</p>
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<p>Construction of Attractant Production and Reporter Pathway</p>
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<p>After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. </p>
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<p>The PRS426 yeast expression vector is used in the expression of our construction. The FUS1 promoter is cloned into the multicloning site via Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned via blunt-end Sma I digest. FUS1 terminator is cloned via Hind III and Xho I digest.</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="https://static.igem.org/mediawiki/2009/9/99/3Figure01.jpg" width=597; height=433 /><br>
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Figure 3. Attractant, reporter and control constructs</p>
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<br><br>
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<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 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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<br>
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<p>Attractant Production Test</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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<br><br>
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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 pRS426-constructed.jpg  " width=550; height=350 /></a>
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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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<a href="http://www.freewebsitetemplates.com"><img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 pRS426.jpg " width=550; height=350 /></a>
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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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<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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  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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  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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<br><br>
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<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li>
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<span> iGEM 2009 <br /> </span>  
<span> iGEM 2009 <br /> </span>  
</div>
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<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="paypal" /></div>
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<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="HKUST" /></div>
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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"