http://2009.igem.org/wiki/index.php?title=Special:Contributions/TonyHo&feed=atom&limit=50&target=TonyHo&year=&month=2009.igem.org - User contributions [en]2024-03-28T12:23:37ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/File:1Figure16.jpgFile:1Figure16.jpg2009-10-22T03:58:45Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/Team:HKUST/Back1Team:HKUST/Back12009-10-22T03:54:48Z<p>TonyHo: </p>
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<div class="contentodS_b"> <h3>a</h3><br />
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<p>GPCRs</p><br />
G protein-coupled receptors (GPCRs) are the main sensors of cells. They respond to ligands such as hormones, neurotransmitters, light, odorants, taste substances, pheromones, and a variety of medicines. GPCRs comprise one of the largest protein superfamilies: it is estimated that there are 1050 and 160 GPCR genes in the genomes of <em>Caenorhabditis elegans</em> and <em>Drosophila melanogaster</em>, corresponding to 5.5% and 1% of their total genes, respectively[1]. Identifying GPCR ligands may lead to the discovery of novel hormones and neurotransmitters, and thus contribute to the development of new medicines. In fact, GPCRs are known to target 30-60% of present medicines[2]. <br><br><br />
GPCRs are seven transmembrane proteins (Fig1). When stimulated by extracellular ligands, the receptors act through heterotrimeric G proteins to regulate intracellular effector proteins. The G proteins are composed of Gα,Gβ and Gγ subunits. In the inactive state, the Gα-subunit is bound to GDP and associated with the Gβ and Gγ dimer. Upon ligand binding, the receptor stimulates the release of GDP from Gα, allowing the binding of GTP to the subunit. Gα-GTP is released from the Gβ and Gγ dimer, and the dissociated subunits regulate the activity of effector proteins such as adenylate cyclase, phospholipase C, mitogen-activated protein kinase (MAP kinase) cascades and ion channels. </p><br><br><br />
<img src="https://static.igem.org/mediawiki/2009/5/57/1Figure00.jpg" width=372; height=169 /></a><br><br />
Fig.1 Model of GPCRs. Adopted from <em>Hinako Suga, Tatsuya Haga, 2007</em>.[3] </p><br><br><br />
Olfactory receptors (OR) are also seven transmembrane proteins. Insect OR gene families have been identified in the fruit fly <em>Drosophila melanogaster</em>, the malaria vector mosquito <em>Anopheles gambiae</em>, the yellow fever vector mosquito <em>Aedes aegypti</em>, the honeybee <em>Apis mellifera</em>, the silkmoth <em>Bombyx mori</em>, and the flour beetle <em>Tribolium castaneum</em>.[4] Though in vertebrates ORs consist largely of GPCRs, in insects there remains doubt whether ORs belong to GPCRs. However, various studies have been carried out on <em>Drosophila</em> and <em>C. elegans</em> G-protein coupled olfactory receptors, and have uncovered much of their structures and functions. <br><br><br />
Based on these facts, in this iGEM project, we choose to study the <em>C.elegans</em> ODR-10 GPCR and the rat RI7 GPCR to demonstrate the working principle in our design. ODR-10 protein is an odorant receptor in <em>C. elegans</em>. It responds to a volatile odorant diacetyl and triggers chemotaxis of <em>C.elegans</em> towards the molecule. RI7 is a rat odorant receptor that has been functionally expressed in yeast. We will show that the </em>C.elegans odorant receptor</em> can also be functionally expressed in yeast and later we could potentially extend this study method to other insect ORs or other GPCRs.</p><br><br><br />
<p>Yeast Pheromone Sensing Pathway</p><br />
<em>Saccharomyces cerevisiae</em> contains two GPCR-based signalling pathways, one that mediates pheromone response during mating and one that senses glucose to regulate filamentous differentiation in diploid cells and invasive growth in haploid cells[5]. There appears to be little crosstalk between these two pathways. By providing a null background, the yeast pheromone response pathway provides a robust and experimentally tractable system to study heterologous GPCRs. <br><br><br />
Haploid <em>S. cerevisiae</em> exists in one of two mating types(a and α), and mating occurs between cells of opposite types and is controlled by the exchange of mating pheromones; a-cells secrete a-factor and express the α-factor receptor (Ste2), whereas α-cells secrete α-factor and express the a-factor receptor (Ste3). Both receptors couple to the same heterotrimeric G protein composed of Gα(Gpa1), Gβ(Ste4) and Gγ (Ste18) subunits. Binding of the mating pheromone to the receptor results in the release of the Gβγ dimer, which then interacts with effectors to bring about conjugation (Fig.2A). A key role of Gβγ is to activate a MAP kinase cascade in which sequential activity of Ste11 (MEK kinase), Ste7 (MAP kinase kinase or MEK) and Fus3 (MAP kinase) leads to activation of a transcription factor (Ste12) that induces gene expression in preparation for mating. Fus3 also activates the cyclindependent kinase inhibitor Far1 to bring about cell cycle arrest[6].<br><br />
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<p>The Expression of Heterologous GPCRs in <em>S. cerevisiae</em></p><br />
<em>S. cerevisiae</em> is chosen in this project as the ideal system to study heterologous GPCR for several reasons(Box 1). </p><br />
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<img src="https://static.igem.org/mediawiki/2009/a/a4/1Figure01.jpg" width=571; height=258 /></a><br />
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To functionally express heterologous GPCRs, we need to do some modifications to the strain to improve the localization of the receptor to the cell membrane and the coupling of the heterologous receptor to the Gα subunit (Fig.2B). </p><br><br><br />
<p>Modifying GPCRs</p><br />
Though some GPCRs may be degraded or retained within intracellular compartments, most GPCRs can be expressed unmodified in yeast. Rat I7 GPCR is one of them, which has been shown to be functionally expressed in the yeast system[8]. Sequence analysis of ORs and GPCRs have shown that the N termini of these receptors are involved in plasma membrane localization, whereas the C termini define the specificity for G protein interaction; the spanning transmembrane domains two (TMII) to seven (TMVII) are the ligand-binding regions. It is possible that a chimeric OR can be expressed using the N termini and C termini of known GPCRs that can be functional expressed, such as RI7, and the ligand binding region of our desired ORs.(For more details, see Experiment Design). </p><br><br><br />
<p>Modifying the G protein</p><br />
The yeast Gα (Gpa1) has been shown to interact with several non-yeast GPCRs. This functional coupling can be improved by replacing Gpa1 with mammalian Gα of the related GPCR, since yeast Gpa1, Ste4 and Ste18 are structurally and functionally similar to mammalian Gα. For example, rat Gαolf and RI7 are both expressed in yeast and RI7 is effectively coupled to Gαolf[8]. <br><br><br />
<p>Developing Reporter Genes</p> <br />
Most yeast GPCR assays use reporter genes under the control of the Ste12-inducible FUS1 promoter. We can either introduce β-galactosidase to provide a quantitative assay suitable for high-throughput analysis; or we can incorporate GFP or luciferase under the FUS1 promoter to fast detect the signal transduction. This method has also been verified in previous studies[8].<br><br><br />
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</html></div>TonyHohttp://2009.igem.org/File:1Figure14.jpgFile:1Figure14.jpg2009-10-22T03:50:45Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure12.jpgFile:1Figure12.jpg2009-10-22T03:49:38Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:Figure11.jpgFile:Figure11.jpg2009-10-22T03:49:14Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure10.jpgFile:1Figure10.jpg2009-10-22T03:48:17Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure09.jpgFile:1Figure09.jpg2009-10-22T03:48:04Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure08.jpgFile:1Figure08.jpg2009-10-22T03:47:46Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure07.jpgFile:1Figure07.jpg2009-10-22T03:46:48Z<p>TonyHo: </p>
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<div></div>TonyHohttp://2009.igem.org/File:1Figure06.jpgFile:1Figure06.jpg2009-10-22T03:46:23Z<p>TonyHo: </p>
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<div class="contentatt_p"> <h3>a</h3><br />
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<p>Attractant Production and Reporter Construct </p><br />
The attractant production pathway consists of three parts: FUS1 promoter, ARO9 gene and FUS1 terminator. These three genes are first derived through PCR from the yeast genome and then cloned into the PRS426 yeast expression vector for further functional assay. <br><br><br />
The reporter construct also contains three parts: FUS1 promoter, EGFP gene and FUS1 terminator. These three genes are first derived through PCR and then cloned into the PRS426 yeast expression vector for further functional assay. <br><br></p><br />
<img src="https://static.igem.org/mediawiki/2009/c/c2/3Figure05.jpg" width=518; height=272 /></a><br><br><br />
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<p> I. Primer Design</p><br />
We have designed several sets of primers for our construction, which are listed in Table 4. These primers are mainly designed for amplification of the FUS1 promoter, EGFP reporter, ARO9 gene and FUS1 terminator. Primer statistics are calculated using NetPrimer. </p><br><br><br />
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<img src="https://static.igem.org/mediawiki/2009/9/9e/3Figure06.jpg" width=584; height=540 /></a><br><br><br />
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<p> II. PCR</p><br />
Three of the four desired genes, FUS1 promoter, FUS1 terminator and ARO9 gene are directly amplified from yeast genomic DNA (strain YPH501). The other gene, EGFP gene, is amplified from the plasmid YTK-6/Tpi/EGFP. <br><br><br />
For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p><br />
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<p> III. Cloning</p><br />
After all the genes are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br><br><br />
The PRS426 yeast expression vector is used in our construction. The FUS1 promoter is cloned into the multiple cloning site by Sac I and Not I digest. The ARO9 gene and the EGFP gene are cloned by blunt-end Sma I digest. FUS1 terminator is cloned by Hind III and Xho I digest. </p><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-22T03:44:51Z<p>TonyHo: </p>
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<p>Construction of Attractant Production and Reporter Pathway</p><br />
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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><br />
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<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure01.jpg" width=597; height=433 /><br><br />
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Figure 3. Attractant, reporter and control constructs</p><br />
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<p>Test of the Reporter System </p><br />
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><br />
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<p>Attractant Production Test</p><br />
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><br />
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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><br />
<img src="https://static.igem.org/mediawiki/2009/9/90/3Figure04.jpg" width=600; height=300 /><br><br><br />
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<p>Further Characterization of the Attractant Production Pathway Function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br><br />
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><br />
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><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-22T03:43:46Z<p>TonyHo: </p>
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<div class="contentatt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant Production and Reporter Pathway</p><br />
<br />
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><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure01.jpg" width=597; height=433 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br><br><br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/c/c7/3Figure02.jpg" width=600; height=280 /><br />
<br><br />
<br />
<p>Attractant Production Test</p><br />
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><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure03.jpg" width=600; height=370 /><br />
<br><br><br />
<br />
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><br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure04.jpg" width=600; height=300 /><br><br><br />
<br />
<p>Further Characterization of the Attractant Production Pathway Function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br><br />
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><br />
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><br />
<br><br><br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-22T03:42:51Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentatt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant Production and Reporter Pathway</p><br />
<br />
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><br />
<br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure01.jpg" width=597; height=433 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br><br><br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure02.jpg" width=600; height=280 /><br />
<br><br />
<br />
<p>Attractant Production Test</p><br />
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><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure03.jpg" width=600; height=370 /><br />
<br><br><br />
<br />
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><br />
<img src="https://static.igem.org/mediawiki/2009/9/99/3Figure04.jpg" width=600; height=300 /><br><br><br />
<br />
<p>Further Characterization of the Attractant Production Pathway Function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br><br />
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><br />
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><br />
<br><br><br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/File:1Figure05.jpgFile:1Figure05.jpg2009-10-22T03:39:58Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:5Figure01.jpgFile:5Figure01.jpg2009-10-22T03:39:27Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:1Figure04.jpgFile:1Figure04.jpg2009-10-22T03:39:10Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:1Figure02.jpgFile:1Figure02.jpg2009-10-22T03:38:37Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/Team:HKUST/Part4Team:HKUST/Part42009-10-22T03:37:47Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentt_p"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/File:1Figure01.jpgFile:1Figure01.jpg2009-10-22T03:37:04Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:1Figure00.jpgFile:1Figure00.jpg2009-10-22T03:36:54Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-22T03:36:16Z<p>TonyHo: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<ul><br />
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/9/91/Figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/a/ac/Figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-22T03:35:22Z<p>TonyHo: </p>
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<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="https://static.igem.org/mediawiki/2009/9/91/Figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2009/9/91/Figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-22T03:32:36Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="https://2009.igem.org/Image:Figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="https://2009.igem.org/Image:Figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/File:3Figure06.jpgFile:3Figure06.jpg2009-10-22T03:31:44Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:3Figure05.jpgFile:3Figure05.jpg2009-10-22T03:30:38Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:3Figure04.jpgFile:3Figure04.jpg2009-10-22T03:28:58Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:3Figure03.jpgFile:3Figure03.jpg2009-10-22T03:26:33Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:3Figure02.jpgFile:3Figure02.jpg2009-10-22T03:24:38Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:3Figure01.jpgFile:3Figure01.jpg2009-10-22T03:22:03Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:Figure03.jpgFile:Figure03.jpg2009-10-22T03:19:05Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:Figure02.jpgFile:Figure02.jpg2009-10-22T03:18:49Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/File:Figure01.jpgFile:Figure01.jpg2009-10-22T03:18:25Z<p>TonyHo: </p>
<hr />
<div></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-22T03:16:23Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="http://ihome.ust.hk/~tonyho/iGem2009/wiki/Group4/figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="http://ihome.ust.hk/~tonyho//iGem2009/wiki/Group4/figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
<br />
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<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-22T03:15:43Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="hhttp://ihome.ust.hk/~tonyho/iGem2009/wiki/Group4/figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="http://ihome.ust.hk/~tonyho//iGem2009/wiki/Group4/figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
<br />
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<br />
<br />
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br><br />
<br />
<br />
<img src="hhttp://ihome.ust.hk/~tonyho//iGem2009/wiki/Group4/figure01.jpg" width=588; height=400 /></a><br><br />
<br />
<br><br />
These constructs will be transformed into yeast and induced by galactose. We will use western blotting to test toxin expressing with an anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill drosophila larvae. The drosophila larvicidal activity is tested by diluting toxin-expressing yeast cells in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br><br><br />
<img src="http://ihome.ust.hk/~tonyho//iGem2009/wiki/Group4/figure02.jpg" width=585; height=505 /></a><br><br />
<br><br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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<span style="color:green"><br />
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<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
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<div class="contentt_p"> <h3>a</h3><br />
</div><br />
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<br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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</ul><br />
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<div id="rightxx"> <br />
<div class="contentt_p"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group4Team:HKUST/Group42009-10-21T15:24:25Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Protein Expression</p><br />
We have designed several constructs to test the binary protein's toxicity to insects as well as to yeast:<br><br />
<br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure01.jpg " width=588; height=400 /></a><br><br />
<br />
These constructs will be transformed into the yeast and induced by galactose. Then we will use western blot to test the expression of toxin by anti-GST antibody. We can estimate the protein level as well as the yeast phenotype: whether they are resistant to the toxin.</p><br />
<br><br />
<p>Drosophila Larvicidal Essay</p><br />
Next we will test the biological function of toxin produced in yeast to kill Drosophila larvae. The Drosophila larvicidal activity is tested by diluting yeast cells expressing the toxin in water and adding them to water containing 10 larvae in each well of a 24-well tissue culture plate (diameter of the well is 1.5 cm)(Figure 1). Mortality is recorded after incubation at 30°C for 48 h. LC50 is determined by Probit analysis[6].</p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group4/figure02.jpg" width=585; height=505 /></a><br><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Back4">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group4">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part4">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Future4">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref4">References</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
</div><br />
<div id="menubottom"><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentatt_p"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Attractant production and reporter Construct </p><br />
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. <br><br />
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><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure05.jpg" width=518; height=272 /></a><br><br />
<br />
Figure 5. Attractant production pathway and Reporter detailed design</p><br />
<br />
<br />
<p> I. Primer Design</p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure06.jpg " width=584; height=540 /></a><br><br />
Table 4 Primer sequences designed for the constructions<br><br />
<br />
*Note: Restriction sites are highlighted in yellow. </p><br />
<br />
<p> II. PCR</p><br />
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. <br><br />
For optimized PCR efficiency and accuracy, Taq polymerase is chosen for amplification PCR reactions. Gradient PCR is carried out for each step to optimize the reaction. </p><br />
<br />
<p> III. Cloning</p><br />
After the all the fragments are successfully derived through PCR, standard cloning procedures are followed to construct an integrated expression vector. <br><br />
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><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
<br />
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<hr />
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<head><br />
<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" /><br />
<link href="http://igem2009hkust.fileave.com/wiki/template/12092009/style.css " rel="stylesheet" type="text/css" /><br />
<title>Salt and Soap template</title><br />
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<div id="leftxx"><br />
<div id="menuxx"><br />
<ul><br />
<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<div id="menubottom"><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"> <br />
<div class="contentatt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant production and Reporter pathway</p><br />
<br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure01.jpg " width=597; height=433 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure02.jpg" width=600; height=280 /><br><br />
<br />
<p>Attractant production test</p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure03.jpg " width=600; height=370 /><br />
<br><br />
<br />
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><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure04.jpg" width=600; height=300 /><br><br />
<br />
<p>Further characterization of the Attractant production pathway function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br />
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><br />
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><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
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<div class="contentatt_d"> <h3>a</h3><br />
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<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant production and Reporter pathway</p><br />
<br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure01.jpg " width=597; height=433 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure02.jpg" width=600; height=280 /><br><br />
<br />
<p>Attractant production test</p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure03.jpg " width=600; height=370 /><br />
<br><br />
<br />
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><br />
<a href="http://www.freewebsitetemplates.com"><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure04.jpg" width=600; height=300 /><br><br />
<br />
<p>Further characterization of the Attractant production pathway function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br />
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><br />
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><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-21T15:13:11Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
<li>Resources</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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<div id="rightxx"> <br />
<div class="contentatt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant production and Reporter pathway</p><br />
<br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure01.jpg " width=597; height=433 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure02.jpg" width=600; height=280 /><br><br />
<br />
<p>Attractant production test</p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure03.jpg " width=600; height=370 /></a><br><br />
<br />
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><br />
<a href="http://www.freewebsitetemplates.com"><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure04.jpg" width=600; height=300 /><br><br />
<br />
<p>Further characterization of the Attractant production pathway function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br />
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><br />
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><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
<br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group3Team:HKUST/Group32009-10-21T15:06:47Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<span style="color:green"><br />
<li>Resources</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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</ul><br />
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</div><br />
<div id="rightxx"> <br />
<div class="contentatt_d"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<br />
<p>Construction of Attractant production and Reporter pathway</p><br />
<br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure01.jpg " width=180; height=80 /><br><br />
<br />
Figure 3. Attractant, reporter and control constructs</p><br />
<br />
<br />
<p>Test of the Reporter System </p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure02.jpg" width=550; height=250 /><br><br />
<br />
<p>Attractant production test</p><br />
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><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure03.jpg " width=400; height=230 /></a><br><br />
<br />
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><br />
<a href="http://www.freewebsitetemplates.com"><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group3/figure04.jpg" width=550; height=350 /><br><br />
<br />
<p>Further characterization of the Attractant production pathway function</p><br />
To further characterize the attractant production pathway function, we can do the following assays. <br><br />
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><br />
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><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back3">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group3">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part3">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result3">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future3">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref3">References</a></li><br />
<br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Result1Team:HKUST/Result12009-10-20T23:37:02Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Team">Our Team</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
<li><a href="https://2009.igem.org/Team:Biosafety">Biosafety</a></li><br />
<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"><br />
<div class="contentodS_re"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Fusion Receptor Construction </p><br />
We have successfully constructed the chimeric receptor cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the C. elegans OR Odr-10 using PCR, and have cloned it into the yeast expression vector pESC-His. Also, we have successfully construct the chimeric receptor tagged with GFP, RI7 tagged with FLAG as well as GFP alone. The agarose gel electrophoresis pictures showing the correct sizes after enzyme digestion of pESC-Fusion-FLAG, pESC-Fusion-GFP, pESC-RI7-FLAG and pESC-GFP are shown in Figure 10.</p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure13.jpg " width=529; height=269 /><br />
</p><br />
<br />
<p>Chimeric Receptor Membrane Localization Assay</p><br />
We have tested the localization of fusion receptor onto the yeast membrane. We have used the constructs pESC-Fusion-GFP and pESC-GFP to carry out the assay. After induction of galactose for 45 minutes, in yeast transformed with pESC-Fusion-GFP, the GFP signal can be seen clearly around the cell membrane, forming a smooth green circle; while no induction or in pESC-GFP cells there is in no such “green circle” (Figure 11). Though there is some GFP expression in the not induced cells and cells of pESC-GFP. This means that the Gal1 promoter is a little leaky, which has been experimentally detected in another study8. After induction for more than 1.5 hours, overexpressed chimeric receptors would aggregate on the membrane, forming intense green patches (Figure 12). The above results show that our chimeric receptor can indeed localize to the yeast membrane. </p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure14.jpg " width=600; height=530 /><br />
<br><br />
<br><br />
<br />
Fig 10. Long time induction of galactose causes overexpression of chimeric receptor so that they aggregate to the membrane forming strong green patches. The left picture shows GFP and the right picture shows live cells. </p><br />
<p>Chimeric Receptor Functioning Assay</p><br />
Part 1<br><br />
We have done receptor functioning assay using diacetyl and hexanal to induce yeasts transformed with pESC-Fusion-FLAG, pESC-RI7-FLAG and pESC vector alone. Though we have not had the reporter pRS426-FUS1P-GFP-FUS1T ready, we could still carry out this assay using the phenomenon of cell cycle arrest at G1 phase as a “reporter”. As a result, we first induced the cells with galactose and ligands diacetyl and hexanal; then we analysed cell DNA contents using FACS assay. The results are shown as follows:<br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure15.jpg " width=600; height=475 /><br />
<br />
<br />
Part 2<br><br />
In this part, we need to first manipulate the yeast strain we are using, which is to knock out FAR1 and GPA1 gene. ……..We have successfully knocked out FAR1 gene and GPA1 gene. <br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure16.jpg " width=584; height=291 /><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref1">References</a></li> <br />
<br />
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</ul><br />
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<div id="rightxx"><br />
<div class="contentodS_re"> <h3>a</h3><br />
</div><br />
<div class="contentxx"><br />
<br />
<p>Fusion Receptor Construction </p><br />
We have successfully constructed the chimeric receptor cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the C. elegans OR Odr-10 using PCR, and have cloned it into the yeast expression vector pESC-His. Also, we have successfully construct the chimeric receptor tagged with GFP, RI7 tagged with FLAG as well as GFP alone. The agarose gel electrophoresis pictures showing the correct sizes after enzyme digestion of pESC-Fusion-FLAG, pESC-Fusion-GFP, pESC-RI7-FLAG and pESC-GFP are shown in Figure 10.</p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure13.jpg " width=529; height=269 /><br />
</p><br />
<br />
<p>Chimeric Receptor Membrane Localization Assay</p><br />
We have tested the localization of fusion receptor onto the yeast membrane. We have used the constructs pESC-Fusion-GFP and pESC-GFP to carry out the assay. After induction of galactose for 45 minutes, in yeast transformed with pESC-Fusion-GFP, the GFP signal can be seen clearly around the cell membrane, forming a smooth green circle; while no induction or in pESC-GFP cells there is in no such “green circle” (Figure 11). Though there is some GFP expression in the not induced cells and cells of pESC-GFP. This means that the Gal1 promoter is a little leaky, which has been experimentally detected in another study8. After induction for more than 1.5 hours, overexpressed chimeric receptors would aggregate on the membrane, forming intense green patches (Figure 12). The above results show that our chimeric receptor can indeed localize to the yeast membrane. </p><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure14.jpg " width=529; height=269 /><br />
<br><br />
<br><br />
<br />
Fig 10. Long time induction of galactose causes overexpression of chimeric receptor so that they aggregate to the membrane forming strong green patches. The left picture shows GFP and the right picture shows live cells. </p><br />
<p>Chimeric Receptor Functioning Assay</p><br />
Part 1<br><br />
We have done receptor functioning assay using diacetyl and hexanal to induce yeasts transformed with pESC-Fusion-FLAG, pESC-RI7-FLAG and pESC vector alone. Though we have not had the reporter pRS426-FUS1P-GFP-FUS1T ready, we could still carry out this assay using the phenomenon of cell cycle arrest at G1 phase as a “reporter”. As a result, we first induced the cells with galactose and ligands diacetyl and hexanal; then we analysed cell DNA contents using FACS assay. The results are shown as follows:<br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure15.jpg " width=600; height=475 /><br />
<br />
<br />
Part 2<br><br />
In this part, we need to first manipulate the yeast strain we are using, which is to knock out FAR1 and GPA1 gene. ……..We have successfully knocked out FAR1 gene and GPA1 gene. <br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure16.jpg " width=584; height=291 /><br />
<br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref1">References</a></li> <br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Part1Team:HKUST/Part12009-10-20T23:31:30Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
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<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
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</div><br />
<div id="rightxx"><br />
<div class="contentodS_p"> <h3>a</h3><br />
</div><br />
<div class="contentxx"> <br />
<br />
<p>Chimeric Receptor Construction</p><br />
The chimeric receptor expression cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the C. elegans OR Odr-10. The receptor sequence is first derived through fusion PCR, and then cloned into the yeast expression vector pESC-His for further localization and functional assay. </p><br />
<br />
I. Primer Design</p><br />
We have designed several sets of primers for parts and BioBrick construction and DNA sequencing. The primer sequences are listed in Table 1. Primer statistics are calculated using NetPrimer. <br><br />
For the chimeric receptor, primers are designed with 10 bp overlapping overhangs at the fusion junctions so that the fragments can anneal in fusion PCR. Two different reverse primers have been designed for the RI7 scaffold primers, one with stop codon incorporated into the sequence, and the other without. These two different alternatives can be chosen for construction of receptors with or without localization tags. In addition, nucleotide sequence in the primers has been modified in a few places to adjust for codon bias among C.elegans, rat and budding yeast. </p><br />
The following diagram illustrates the schematic of the main primer design for the chimeric receptor. <br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure10.jpg " width=569; height=184 /></p><br />
<br><br />
<br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure11.jpg " width=600; height=450 /><br />
</p><br />
<br />
II. PCR</p><br />
The DNA fragments coding for the RI7 localization scaffold and the Odr-10 ligand-binding pocket are first amplified separately from respective cDNA template (pHeI4 for RI7 and pPD9S.77 for Odr-10) via PCR. These fragments are then fused together via fusion PCR. <br><br />
In fusion PCR, reaction conditions and reactant concentrations must be carefully controlled to obtain satisfactory yields. DNA fragment fusion must be carried out in a stepwise process, by fusing RI7-N and Odr-10 first (using P1 and P5), followed by annealing this fused sequence to the RI7-C fragment (using P1 and P2). PCR cleanup is essential in the procedures, preferably by gel purification, since residual primers may affect the reaction in the next step, leading to amplification of the original template instead of desired fusion reaction. <br><br />
In the reaction, the two fragment templates are allowed to go through 1 to 2 PCR cycles without primers in order to create a fused template for amplification. The amplification primers are then added to the reaction mixture to allow for amplification of the whole fused sequence. <br><br />
<br />
III. Cloning</p><br />
After the chimeric receptor sequence is successfully derived through fusion PCR, standard cloning procedures are followed to construct a receptor expression cassette. <br><br />
The pESC yeast epitope tagging vector pESC-HIS is used in the expression cassette construction. The chimeric receptor insert is cloned into MCS1 of the vector (under GAL10 promoter; with FLAG epitope tag) between the restriction sites EcoRI and NotI. The RI7 control construct can also be generated in the same way. <br><br />
A GFP-tagged receptor can be generated by cloning the GFP tag into a constructed receptor expression vector, between the MCS1 sites of SpeI and SacI, replacing the FLAG epitope (Figure 9). <br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure12.jpg " width=600; height=350 /> <br />
<br><br />
<br />
<br><br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref1">References</a></li><br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Part1Team:HKUST/Part12009-10-20T23:29:19Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Main Parts</li><br />
</span><br />
</b><br />
<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
<br />
<b><br />
<span style="color:green"><br />
<li>Resources</li><br />
</span><br />
</b><br />
<br />
<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Resourses">Other Resources</a></li> <br />
</ul><br />
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<li><a href="https://2009.igem.org/Team:Gallery">Gallery</a></li><br />
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<li><a href="https://2009.igem.org/Team:Acknowledgement">Acknowledgement</a></li><br />
</ul><br />
</div><br />
</div><br />
<div id="rightxx"><br />
<div class="contentodS_p"> <h3>a</h3><br />
</div><br />
<div class="contentxx"> <br />
<br />
<p>Chimeric Receptor Construction</p><br />
The chimeric receptor expression cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the C. elegans OR Odr-10. The receptor sequence is first derived through fusion PCR, and then cloned into the yeast expression vector pESC-His for further localization and functional assay. </p><br />
<br />
I. Primer Design</p><br />
We have designed several sets of primers for parts and BioBrick construction and DNA sequencing. The primer sequences are listed in Table 1. Primer statistics are calculated using NetPrimer. <br><br />
For the chimeric receptor, primers are designed with 10 bp overlapping overhangs at the fusion junctions so that the fragments can anneal in fusion PCR. Two different reverse primers have been designed for the RI7 scaffold primers, one with stop codon incorporated into the sequence, and the other without. These two different alternatives can be chosen for construction of receptors with or without localization tags. In addition, nucleotide sequence in the primers has been modified in a few places to adjust for codon bias among C.elegans, rat and budding yeast. </p><br />
The following diagram illustrates the schematic of the main primer design for the chimeric receptor. <br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure10.jpg " width=569; height=184 /></p><br />
<br><br />
<br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure11.jpg " width=600; height=450 /><br />
</p><br />
<br />
II. PCR</p><br />
The DNA fragments coding for the RI7 localization scaffold and the Odr-10 ligand-binding pocket are first amplified separately from respective cDNA template (pHeI4 for RI7 and pPD9S.77 for Odr-10) via PCR. These fragments are then fused together via fusion PCR. <br><br />
In fusion PCR, reaction conditions and reactant concentrations must be carefully controlled to obtain satisfactory yields. DNA fragment fusion must be carried out in a stepwise process, by fusing RI7-N and Odr-10 first (using P1 and P5), followed by annealing this fused sequence to the RI7-C fragment (using P1 and P2). PCR cleanup is essential in the procedures, preferably by gel purification, since residual primers may affect the reaction in the next step, leading to amplification of the original template instead of desired fusion reaction. <br><br />
In the reaction, the two fragment templates are allowed to go through 1 to 2 PCR cycles without primers in order to create a fused template for amplification. The amplification primers are then added to the reaction mixture to allow for amplification of the whole fused sequence. <br><br />
<br />
III. Cloning</p><br />
After the chimeric receptor sequence is successfully derived through fusion PCR, standard cloning procedures are followed to construct a receptor expression cassette. <br><br />
The pESC yeast epitope tagging vector pESC-HIS is used in the expression cassette construction. The chimeric receptor insert is cloned into MCS1 of the vector (under GAL10 promoter; with FLAG epitope tag) between the restriction sites EcoRI and NotI. The RI7 control construct can also be generated in the same way. <br><br />
A GFP-tagged receptor can be generated by cloning the GFP tag into a constructed receptor expression vector, between the MCS1 sites of SpeI and SacI, replacing the FLAG epitope (Figure 9). <br><br />
<br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure11.jpg " width=600; height=450 /> <br />
<br><br />
<br />
<br><br />
<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref1">References</a></li><br />
<br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Part1Team:HKUST/Part12009-10-20T23:28:27Z<p>TonyHo: </p>
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<div class="contentodS_p"> <h3>a</h3><br />
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<p>Chimeric Receptor Construction</p><br />
The chimeric receptor expression cassette contains the N- and C- terminals of the rat OR RI7, flanking the TM2-TM7 ligand-binding domain of the C. elegans OR Odr-10. The receptor sequence is first derived through fusion PCR, and then cloned into the yeast expression vector pESC-His for further localization and functional assay. </p><br />
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I. Primer Design</p><br />
We have designed several sets of primers for parts and BioBrick construction and DNA sequencing. The primer sequences are listed in Table 1. Primer statistics are calculated using NetPrimer. <br><br />
For the chimeric receptor, primers are designed with 10 bp overlapping overhangs at the fusion junctions so that the fragments can anneal in fusion PCR. Two different reverse primers have been designed for the RI7 scaffold primers, one with stop codon incorporated into the sequence, and the other without. These two different alternatives can be chosen for construction of receptors with or without localization tags. In addition, nucleotide sequence in the primers has been modified in a few places to adjust for codon bias among C.elegans, rat and budding yeast. </p><br />
The following diagram illustrates the schematic of the main primer design for the chimeric receptor. <br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure10.jpg " width=569; height=184 /></p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure11.jpg " width=600; height=450 /><br />
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II. PCR</p><br />
The DNA fragments coding for the RI7 localization scaffold and the Odr-10 ligand-binding pocket are first amplified separately from respective cDNA template (pHeI4 for RI7 and pPD9S.77 for Odr-10) via PCR. These fragments are then fused together via fusion PCR. <br><br />
In fusion PCR, reaction conditions and reactant concentrations must be carefully controlled to obtain satisfactory yields. DNA fragment fusion must be carried out in a stepwise process, by fusing RI7-N and Odr-10 first (using P1 and P5), followed by annealing this fused sequence to the RI7-C fragment (using P1 and P2). PCR cleanup is essential in the procedures, preferably by gel purification, since residual primers may affect the reaction in the next step, leading to amplification of the original template instead of desired fusion reaction. <br><br />
In the reaction, the two fragment templates are allowed to go through 1 to 2 PCR cycles without primers in order to create a fused template for amplification. The amplification primers are then added to the reaction mixture to allow for amplification of the whole fused sequence. <br><br />
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III. Cloning</p><br />
After the chimeric receptor sequence is successfully derived through fusion PCR, standard cloning procedures are followed to construct a receptor expression cassette. <br><br />
The pESC yeast epitope tagging vector pESC-HIS is used in the expression cassette construction. The chimeric receptor insert is cloned into MCS1 of the vector (under GAL10 promoter; with FLAG epitope tag) between the restriction sites EcoRI and NotI. The RI7 control construct can also be generated in the same way. <br><br />
A GFP-tagged receptor can be generated by cloning the GFP tag into a constructed receptor expression vector, between the MCS1 sites of SpeI and SacI, replacing the FLAG epitope (Figure 9). <br><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure11.jpg " width=600; height=350 /> <br />
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<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
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</html></div>TonyHohttp://2009.igem.org/Team:HKUST/Group1Team:HKUST/Group12009-10-20T23:22:34Z<p>TonyHo: </p>
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<li><a href="https://2009.igem.org/Team:HKUST/Project">Project Description</a></li><br />
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<span style="color:green"><br />
<li>Main Parts</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/OdorantSensing">Odorant Sensing</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/AttractantProduction">Attractant Production</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/ToxinProduction">Toxin Production</a></li><br />
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<li>Resources</li><br />
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<li><a href="https://2009.igem.org/Team:HKUST/Lab Notebook">Lab Notebook</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Parts">Parts Submitted </a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Protocols">Protocol List</a></li> <br />
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<div class="contentodS_d"> <h3>a</h3><br />
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<p>Construction of Receptor Expression Cassette</p><br />
To functionally express C. elegans Odr10 receptor, we have designed an expression cassette. Based on the fact that rat I7 (RI7) receptor has been successfully expressed in the yeast system and it couples well to Gαolf subunit, we have designed such a chimeric receptor with the RI7 N (amino acids 1-61 of RI7) and C termini (animo acids 295-327 of RI7) fused with Odr10 transmambrane domains two to seven (TM2 – 7, amino acids 48-305 of Odr10, starting from the fourth amino acid of Odr10 TM2) [i] (figure 3). At the N termini side, they are fused at the junctions PMYFF (RI7) and YLMAFF (Odr10) because these two sequences are conserved in both receptor junction sites (Figure 4). They are cloned into the multiple cloning site of pESC-HIS expression vector under the Gal1 promoter. They are also tagged with FLAG or GFP at the C termini for protein expression detection or localization test. Given that we have retained the N and C termini of RI7, localization and subsequent coupling should not be perturbed.</p><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure03.jpg " width=584; height=372 /></a><br><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure04.jpg " width=584; height=308 /></a><br><br />
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<p>Test of Chimeric Protein Localization</p><br />
To test the localization of chimeric proteins, we have made the construct pESC-Fusion-GFP, which is the chimeric protein with the GFP tag at the C termini in the expression vector pESC-HIS, as well as the negative control pESC-GFP, which is only the GFP at the same site in the expression vector pESC-HIS (Figure 5). We choose to tag GFP at the C termini because N termini of the fusion protein is important for membrane localization while C termini is important for coupling. Since we want to ensure its proper localization, it is better to fuse GFP at the C termini. </p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure05.jpg " width=612; height=358 /></a><br />
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After transformation and selection, we would induce the transformants with galactose at the exponential stage of yeast growth. After some time, we would harvest cells for the fluorescence microscopy test. We would expect to see the cells transformed with pESC-Fusion-GFP to have strong green fluorescence surrounding the cell, forming a green circle; while the negative control does not. In that case, we could say that the fusion protein is able to localize in the yeast membrane. </p><br />
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<p>Test of Chimeric Protein Function</p><br />
To test the functional sensing and coupling to Gαolf or Gpa1, we have made several constructs: <br><br />
<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure06.jpg" width=573; height=575 /><br />
</p><br />
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There are two parts of the fuctional coupling test:</p><br />
Part 1<br><br />
First we test whether our chimeric protein could couple to Gpa1. <br><br />
The yeasts would be transformed with construct (1)+(4) or (2)+(4) or (5)+(4), respectively. After induction with galactose to express the receptors, we would add in the ligands diacetyl (for Odr10) and hexanal (for RI7). After some time of ligands binding, we would expect to see that the functional receptors could couple to Gpa1 and then trigger downstream FUS1-promoter-driven expression of GFP, together with cell cycle arrest at G1 phase (figure 6). The expression of GFP could be viewed by fluorescence microscopy; the cell cycle arrest could be confirmed by fluorescence-activated cell sorting (FACS). We might expect to see the fusion receptor and RI7 response to diacetyl and hexanal, respectively, to have GFP and cell cycle arrest. In that case we could say that fusion receptor could functionally couple to Gpa1 and start downstream signalling. <br><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure07.jpg" width=600; height=515 /><br />
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Fig 6 Ligands sensing functional assay showing control experiments. (a) is yeast transformed with pESC-Fusion-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and diacetyl are added; (b) is yeast transformed with pESC-RI7-FLAG & pRS426-FUS1P-GFP-FUS1T, and we would expect to see GFP and cell cycle arrest only when both galactose and hexanal are added; (c) is yeast transformed with pESC empty plasmid and pRS426-FUS1P-GFP-FUS1T, and we would expect to see no GFP and cell cycle arrest. These cells are analyzed with fluorescence microscopy and FACS. </p><br />
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Part 2<br><br />
Second, we test whether our chimeric receptor could couple to Gαolf for optimized signalling transduction as well as constructing a testable yeast system for odorant sensing. <br><br />
Gene deletion<br><br />
Before testing, yeast strain needs to be manipulated: we need to knock out the FAR1 gene, which encodes a protein controlling cell cycle arrest upon activation of MAPK pathway, so that the yeasts will still be viable after ligand binding; and also endogenous GPA1 gene, so that we could replace it with Gαolf. Due to the second messenger activity of the free G protein βγ subunit, haploid S. cerevisiae strains containing a null mutation of GPA1 undergo constitutive pheromone response of G1 arrest resulting in non-viability[ii]. So, we need to first knock out cell cycle regulatory gene FAR1 and afterwards, GPA1.To knock out these two genes, we have adopted the PCR-based tagging of yeast genes method, introduced by Janke C., el al, 2004[iii]. <br><br />
To test the successful deletion, we will use both PCR confirmation (figure 7) and also phenotype observation after adding α-factor (no mating phenotype). </p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure08.jpg" width=585; height=465 /><br />
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Next, we need to transform Gαolf into the double knock-out strain to check for positive transformants. It has been reported that Gαolf could not only complement for a GPA1 deficiency but also can functionally couple to the pheromone receptor STE211. Thus we have developed assays to select functional Gαolf (figure8). </p><br />
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<img src="http://igem2009hkust.fileave.com/wiki/Group1/figure09.jpg" width=607; height=413 /><br />
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After confirmation of functional Gαolf, we would use this strain to test its ligand binding using the same method shown in figure 6. However, this time we would expect to see no cell cycle arrest in any case since FAR1 has been deleted. We could expect the ligand binding assay this time could give an optimized coupling and signalling transduction. </p><br />
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<p>Further Characterization of the Chimeric Protein Function</p><br />
To further characterize how this chimeric receptor function, we can do the following assays: <br><br />
First, we can test the how it responses to different ligand concentrations. According to literature, C. elegans Odr10 responses strongest to 100μM diacetyl[i], and we can then compare the intensity of GFP production under a series of diacetyl concentrations. <br><br />
Second, we can test the ligand specificity of the chimeric protein. According to literature, Odr10 responses to volatile molecule diacetyl, as well as non-volatile molecule pyruvic acid and citric acid13. We can also test whether it can response to other volatile molecules by setting up similar assays shown in figure 6. <br><br />
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<br><br />
<li><a href="https://2009.igem.org/Team:HKUST/Back1">Background</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Group1">Experimental Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Part1">Parts Design</a></li> <br />
<li><a href="https://2009.igem.org/Team:HKUST/Result1">Experimental Results</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Future1">Future Work</a></li><br />
<li><a href="https://2009.igem.org/Team:HKUST/Ref1">References</a></li><br />
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