Coding

Part:BBa_K1603000

Designed by: John Hellgren   Group: iGEM15_Chalmers-Gothenburg   (2015-09-18)
Revision as of 21:35, 18 September 2015 by Jannie (Talk | contribs)

Fusion GPCR STE2MAM2

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. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 408
  • 1000
    COMPATIBLE WITH RFC[1000]


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


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).

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

ChalmersGothenburgC4230.jpg
Figure 2. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgC40.jpg
Figure 3. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgWT230.jpg
Figure 4. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

ChalmersGothenburgWT0.jpg
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 weak promoter pFUS1 [2] 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

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