Team:TU-Delft/part3
From 2012.igem.org
Content
IntroductionParts
Results
Conclusions
Recommendations
Introduction
By combining the olfactory receptor and the FUS1pr-EGFP reporter, a complete yeast olfactory system is obtained. If the corresponding ligand binds to the receptor the FAR1 promoter is turned on and the EGFP is expressed. This EGFP signal can be read out by a fluorescence meter. If the olfactory system will be implemented as a diagnostics tool in developing countries, the EGFP reporter should be changed by a visible reporter.
Besides the induction of the FUS1 promoter the cells also go in growth arrest mediated by the FAR1 promoter. However it is undesirable that the cells stop growing once they respond to a ligand. Therefore it is needed to knock out the FAR1 promoter.
Parts
The receptor constructs and the reporter constructs are combined to have one complete olfactory system. The following biobricks are created:
BBa_K775005
BBa_K775006
BBa_K775007
BBa_K775008
Results
Knock out FAR1
Setup
Outcome
For making a functional knockout, yeast strains BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YJL157c::kanMX4 and BY4741; Mat a; his3D1; leu2D0; met15D0; ura3D0; YHR005c::kanMX4 were used (Euroscarf). Also knockout cassette pUG72 is used (Euroscarf). The LoxP-Ura-LoxP is elongated using the pFx polymerase protocol. The PCR program and primer sequences in table 1 yielded a product which is put on a gel shown in figure 1. Here a band can be recognized between 2000 and 1500 nucleotides, which corresponds to the 1669 nucleotide PCR product.Table 1 PCR program for elongation of knockout cassette for knocking out Far1 and primers used for elongation.
Repeats | Temperature | Duration | 5x | 95 | Melting | 2:00 |
---|---|---|---|
51 | Annealing | 1:00 | |
68 | Elongation | 2:00 | |
25x | 95 | Melting | 2:00 |
61 | Annealing | 1:00 | |
68 | Elongation | 2:00 |
FAR1 ko fw ACACAAAGTCTATAGATCCACTGGAAAGCTTCGTGGGCGTAAGAAGGCAATCTATTAATGCAGCTGAAGCTTCGTACGC |
FAR1 ko rv GAAAAAAAAAAAAGGAAAAGCAAAAGCCTCGAAATACGGGCCTCGATTCCCGAACTACTAGCATAGGCCACTAGTGGATCTG |