Team:HKUST/Result1

Fusion Receptor Construction

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.

Fig 10. Agarose gel electrophoresis pictures showing the correct size after enzyme digestion of pESC-Fusion-FLAG, pESC-Fusion-GFP, pESC-RI7-FLAG and pESC-GFP.

Chimeric Receptor Membrane Localization Assay

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.

Fig 9. Membrane localization test using fluorescence microscopy. The upper picture shows GFP and the lower picture shows the live cells. (a) is the yeast transformed with pESC-Fusion-GFP induced with galactose for 45mins. (b) is the yeast transformed with pESC-Fusion-GFP not induced with galactose. (c) is the yeast transformed with pESC- GFP induced with galactose for 45mins.

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.

Chimeric Receptor Functioning Assay

Part 1
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:
Part 2
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.

Background

Experimental Design

Parts Design

Experimental Results

Future Work

Reference