Team:HKUST/Result1

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 1). 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 1(b)). The above results show that our chimeric receptor can indeed localize to the yeast membrane.

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

References