Part:BBa_K1603001
pTEF1-pSUC2
The high expression pTEF1 promoter connected to pSUC2 promoter. Can be used for induced high expression of any coding part in Saccharomyces cerevisiae at low ATP levels.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 167
Expression of mRFP (monomeric Red Fluorescent Protein) was used to analyze expression levels. To evaluate the effect of connecting pTEF1 to pSUC, two different versions were made: pTEF1-pSUC2-mRFP (TEFSUC) and pSUC2-mRFP (SUC). Both constructs were integrated in S.cerevisiae CEN.PK2 and the fluorescent was compared with wild type CEN.PK2(WT). in expression levels of serially connecting these two promoters, mRFP (monomeric Red Fluorescent Protein) was added downstream of pSUC2 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 quantitative difference in expression.
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