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Team:HKUST/Group3
From 2009.igem.org
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Construction of Attractant Production and Reporter Pathway
To functionally express the yeast ARO9 gene and EGFP gene, we have designed reporter and attractant production constructs. Since yeast endogenous FUS1 promoter can sense the downstream signal generated by the ligand-binding receptor, and the yeast endogenous FUS1 terminator can stop the transcription activated by the promoter, we have designed a pathway that puts the ARO9 gene (with the original promoter deleted) and the EGFP gene under the control of the FUS1 promoter. We have also designed control constructs for later experiments. The whole system is cloned into the multiple cloning site of the pRS426 yeast expression vector.
Figure 3. Attractant, reporter and control constructs
Test of the Reporter System
The yeast strain YPH501 with ARO9 gene deleted would be transformed with pRS426-FUS1P-EGFP-FUS1T and pRS426-EGFP respectively. After α factor binding, we would expect to see that the MAPK pathway is activated, leading to the downstream FUS1-promotor-driven expression of GFP. The expression of GFP could be viewed by fluorescence microscopy.
Attractant Production Test
We will test our attractant production pathway and the attractant effect on drosophila using the Cage Bioassays method given by J. Zhu, et al, 2003.(Figure 4)
By comparing these six conditions, we can decide whether the attractant pathway under the FUS1 promoter can respond to signals generated by the receptor and effectively produce attractant molecules (Table 2).
Further Characterization of the Attractant Production Pathway Function
To further characterize the attractant production pathway function, we can do the following assays.First, we can use the HPLC or other analytical chemical methods to analyze the attractant concentration in the yeast extract and find the best attractant concentration towards drosophila.
Second, we can change the strain of the yeast to test the attractiveness differences between strains and find the best strain for attractant production.
iGEM 2009
