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 S.cerevisiae to detect the Pheromone P-factor through the Pheromone pathway.
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 analyze the effect in expression levels of serially connecting these two promoters, pTEF1-pSUC2 was connected to mRFP and transformed into the genome of S.cerevisiae. The first sample of TEFSUC, SUC and WT was cultivated for 2 hours in YPD. The results from fluorescent microscopy are shown in figure 1-3.
Figure 1. TEFSUC sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 2. SUC sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 3. WT sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
3 hours of cultivation in YPD (after overnight preculture) gives no visible RFP fluorescence. A reason for this could be that 2 hours is not enough to give a significant drop in energy levels to relieve the repression of pSUC2.
A new sample of TEFSUC, SUC and WT was cultivated for 6 hours in YPD. The results from fluorescent microscopy are shown in figure 4-6.
Figure 4. TEFSUC sample 2 cultivated for 6 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 5. SUC sample 2 cultivated for 6 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 6. WT sample 2 cultivated for 6 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Now there is a clear difference between TEFSUC and SUC. TEFSUC gives several highly fluorescent cells while SUC only shows slightly higher fluorescent compared to WT. This indicates that the repression of pSUC2 is reduced which allows expression of mRFP through the high expression promoter pTEF1.
Another fluorescence measurement was performed on the same sample after 23 hours of cultivation. The results from fluorescent microscopy are shown in figure 7-9.
Figure 7. TEFSUC sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 8. SUC sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
Figure 9. WT sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.
This demonstrates that the concept of serially could work, but further evaluation could include measuring of fluorescent levels to determine the actual difference in expression rates.
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