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Team:HKUST/Result1
From 2009.igem.org
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Fusion Receptor Construction
We have successfully constructed the chimeric receptor using PCR, and have cloned it into the yeast expression vector pESC-His. Also, we have successfully constructed the chimeric receptor tagged with GFP, RI7 tagged with FLAG, as well as GFP alone inserted into the vector. Figure 10 shows the agarose gel electrophoresis pictures, highlighting the correct sizes after enzyme digestion of pESC-Fusion-FLAG and pESC-RI7-FLAG.
Chimeric Receptor Membrane Localization Assay
We have tested the localization of the chimeric 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, yeast transformed with pESC-Fusion-GFP shows clear GFP signal around the cell membrane, forming a smooth green halo; whilst non-induced cells show no such green halo (Figure 11). Nevertheless, there is some GFP expression in the non-induced cells, which means that the GAL1 promoter is somewhat leaky. This has been experimentally detected in another study[8]. After induction for more than 1.5 hours, over-expression of chimeric receptors results in stronger GFP intensity (Figure 11(b)). The above results show that our chimeric receptor can indeed localize to the yeast membrane.
Chimeric Receptor Functional Assay
Part 1We have performed receptor functional assay, using diacetyl and hexanal to induce yeasts transformed with pESC-Fusion-FLAG, pESC-RI7-FLAG and pESC empty vector. Although 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; then we used the ligands diacetyl(5mM) and hexanal(5mM) to induce yeast cells; finally we analyzed cell DNA contents by FACS assay. The results are shown as follows (Figure 12).
As we can see, compared with the control in which no ligands were added, RI7 FACS shows that the majority of cells stop at G1 phase after 2 hours of induction with its ligand hexanal; the chimeric receptor FACS also shows cell cycle arrest at G1 phase for the majority of the population after induction with diacetyl for 2 hours. We could not see the whole population arrested at G1 phase, which is as we have expected because we are using the yeast endogenous GPA1 subunit to couple with RI7-C terminus, and this coupling is not as efficient as Gαolf, according to literature. We can also conclude that RI7 and our chimeric receptor have similar efficiency in sensing and coupling, because the number of cells arrested at G1 phase and the response time to the ligands (before 2 hours) are similar for both.
In sum, we can say that our chimeric receptor is functioning as a ligand sensing receptor for the volatile molecule diacetyl.
Part 2
In this part, we need to first manipulate the yeast strain we are using by knocking out the FAR1 and GPA1 genes.
First we need to knock out the FAR1 gene, so that cells will not arrest at G1 phase after ligand binding. The following result shows successful FAR1 deletion (Figure13). After α factor induction, wild type cells display mating phenotype “shmoo”; FAR1 knock-out strain only shows normal yeast phenotypes “budding”.
Then we proceeded to delete the GPA1 gene from the △FAR1 strain using the same method. This time, as there are no particular phenotypes of the deletion strain, we used PCR to confirm the successful knock-out strain. By carefully designing different sets of primers, we were able to identify the colony that showed proper bands as we have expected.
To conclude, we have successfully knocked out the FAR1 gene and the GPA1 gene. So far, we have made our desired strain to functionally express our engineered odorant sensing chimeric receptor.
iGEM 2009
