Difference between revisions of "Part:BBa K1603000"

Latest revision as of 01:38, 19 September 2015

Fusion GPCR STE2MAM2

Summary

Fusion GPCR of the non-cytoplasmic N-terminal signal peptide from STE2 (Saccharomyces cerevisiae) and Pheromone P-factor receptor MAM2 (Schizosaccharomyces pombe) without its signaling peptide. Allows in vivo detection of the Pheromone P-factor from S.pombe through the Pheromone pathway in S.cerevisiae.

Experimental documentation

To evaluate the detection properties of this part, STE2MAM2 was connected to the promoter pTDH3 and the reporter gene mRFP (monomeric Red Fluorescent Protein) was connected to the pFUS1, which is activated at the end of the pheromone pathway [1].

To test the detection system in Saccharmyces cerevisiae, h+ pheromones from S. pombe had to be extracted. S. pombe L972 +h was therefore grown in YPD media at 30 ˚C for 3 weeks. The pheromones was extracted by pelleting the cells and filtrating the supernatant through a filter with 0.2 μM pore size. To increase the concentration of the h+ phermones, the solution was run in a concentrator with a cold trap for 5,5 h. The concentration was increased by approximately 20 times.

A test of the detection system was then performed accordingly:

1. A colony of S. cerevisiae CEN.PK2 integrated with construct 4 was inoculated in 5 ml YPD media overnight. Wild type CEN.PK2 and a strain expressing RFP was used as negative and positive control.

2. The OD was measured after an overnight pre-culture and the cells was centrifuged at 1100 rcf for 5 min.

3. The pellet was dissolved in 5 ml YPD media and transferred to a shake flask. The solution was diluted with YPD to a OD of 0,4. The cells were inoculated at 30 ˚C for 2h.

4. 0, 50, 115 and 230 μL concentrated pheromones were added to 1 ml cell suspensions of the negative control and the colony with C4. No pheromones were added to the positive control. The cells were once again incubated at 30 ˚C for 2h to allow expression of RFP.

5. The cells were pelleted and washed with once with distilled water. The pellet was dissolved in 70μl distilled water and 3 μL of the solution was used for examining the detection system using a fluorescence microscope.

6. 1 second exposure time was used for RFP measurement. GFP exposure was used as a measure of inviable cells.

The fluorescent microscopy pictures from the detection test with CEN.PK2 containing construct 4 (C4) are shown in figure 1-5. Samples were made with different amounts of concentrated P-factor and compared with wild type CEN.PK2 (WT).

Figure 1. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 2. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 3. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 4. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 5. WT without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Only the C4 samples containing the highest amount of P-factor showed a few fluorescent cells, which is promising for our concept. The weak signal can be explained by the fact that constructs containing the amplification system through dCas9-vp64 could not be completed. This forces the detection system to rely on the presumably weak promoter pFUS1 to express mRFP. This can result in a weak fluorescent signal when the P-factor is detected.

References

[1] Bardwell, L. (2005). A walk-through of the yeast mating pheromone response pathway (vol 25, pg 1465, 2004). Peptides, 26(2), 337-337. doi:10.1016/j.peptides.2004.10.001 Sequence and Features