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Team:HKUST/Group3
From 2009.igem.org
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To functionally express the yeast ARO9 gene and EGFP gene, we have designed a reporter and an attractant production constructs. Based on the fact that yeast endogenous FUS1 promoter can sense the signal generated by the receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed such a pathway that puts ARO9 gene (with the original promoter deleted) and EGFP gene under the control of the FUS1 promoter. We have also have control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector. </p> | 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> | ||
| - | + | <img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design1.jpg " width=180; height=80 /></a><br> | |
(1) pRS426-FUS1P-ARO9-FUS1T, which is ARO9 gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p> | (1) pRS426-FUS1P-ARO9-FUS1T, which is ARO9 gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p> | ||
| - | + | <img src="http://igem2009hkust.fileave.com/wiki/Group3/Gp3 design2.jpg " width=180; height=80 /></a><br> | |
(2) pRS426-FUS1P-EGFP-FUS1T, which is EGFP gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p> | (2) pRS426-FUS1P-EGFP-FUS1T, which is EGFP gene under the control of FUS1 promoter, cloned into the plasmid pRS426.</p> | ||
| - | + | <img src="http://igem2009hkust.fileave.com/wiki/Group3/pRS426-EGFP.jpg" width=150; height=80 /></a><br> | |
(3) pRS426-EGFP, which is EGFP gene without the FUS1 promoter, cloned into the plasmid pRS406. </p> | (3) pRS426-EGFP, which is EGFP gene without the FUS1 promoter, cloned into the plasmid pRS406. </p> | ||
Figure 3. Attractant, reporter and control constructs</p> | Figure 3. Attractant, reporter and control constructs</p> | ||
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The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T, pRS426-EGFP, respectively. After α factor binding, we would expect to see that the MAPK pathway is activated and then the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy. </p> | 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> | ||
| - | + | <img src="http://igem2009hkust.fileave.com/wiki/Group3/Table 1 Reporter functional assay and expected results.jpg " width=550; height=250 /></a><br> | |
Table 1 Reporter functional assay and expected results. (a) is the YPH501 strain deleted with ARO9 gene and transformed with pRS426-FUS1P-EGFP-FUS1T; (b) is the same strain transformed with pRS426-EGFP. </p> | Table 1 Reporter functional assay and expected results. (a) is the YPH501 strain deleted with ARO9 gene and transformed with pRS426-FUS1P-EGFP-FUS1T; (b) is the same strain transformed with pRS426-EGFP. </p> | ||
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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> | 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> | ||
| - | + | <img src="http://igem2009hkust.fileave.com/wiki/Group3/test.jpg " width=400; height=230 /></a><br> | |
Figure 4, The cage bioassay to show the attractant effect. </p> | Figure 4, The cage bioassay to show the attractant effect. </p> | ||
Revision as of 07:05, 11 October 2009
Welcome
Construction of Attractant production and Reporter pathway
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.
(1) pRS426-FUS1P-ARO9-FUS1T, which is ARO9 gene under the control of FUS1 promoter, cloned into the plasmid pRS426.
(2) pRS426-FUS1P-EGFP-FUS1T, which is EGFP gene under the control of FUS1 promoter, cloned into the plasmid pRS426.
(3) pRS426-EGFP, which is EGFP gene without the FUS1 promoter, cloned into the plasmid pRS406. Figure 3. Attractant, reporter and control constructs
Test of the Reporter System
The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T, 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.
Table 1 Reporter functional assay and expected results. (a) is the YPH501 strain deleted with ARO9 gene and transformed with pRS426-FUS1P-EGFP-FUS1T; (b) is the same strain transformed with pRS426-EGFP.
Attractant production test
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)
Figure 4, The cage bioassay to show the attractant effect. 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).
Table 2. Different traps and expected results
Further characterization of the Attractant production pathway function
To further characterize the attractant production pathway function, we can do the following assays.First, we can use the HPLC or some other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards Drosophilae.
Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production.
iGEM 2009
