Part:BBa_K1603000
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 408
- 1000COMPATIBLE WITH RFC[1000]
To evaluate the detection properties of this part, STE2MAM2 was connected to the promoter pTDH3
The fluorescent microscopy pictures from the detection test with CEN.PK2 containing construct 4 (C4) are shown in figure 3-6. Samples were made with different amounts of concentrated P-factor and compared with wild type CEN.PK2 (WT).
Figure 3. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 4. C4 with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 5. C4 without P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 6. WT with 230 µl concentrated P-factor. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 7. 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 [1] to express mRFP. This can result in a weak fluorescent signal when the P-factor is detected.
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