Difference between revisions of "Part:BBa K1603001"

Revision as of 03:39, 19 September 2015

pTEF1-pSUC2

Summary

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. Improves the function of pTEF1 by adding repression at high levels of ATP and induction at low levels of ATP.

Experimental documentation

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 and pSUC2-mRFP. Both constructs were integrated into the genome of S.cerevisiae CEN.PK2. The fluorescence of the clone with integrated pTEF1-pSUC2-mRFP (TEFSUC) and the clone with pSUC2-mRFP (SUC) was compared with wild type CEN.PK2 (WT). The first sample of TEFSUC, SUC and WT was cultivated for 2 hours in YPD. The results from fluorescent microscopy are shown in figure 1-4.

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

Figure 2. TEFSUC sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 3. SUC sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 4. WT sample 1 cultivated for 2 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

2 hours of cultivation in YPD (after overnight preculture) showed 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 5-8.

Figure 5. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 6. TEFSUC sample 2 cultivated for 6 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 7. SUC sample 2 cultivated for 6 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 8. 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 9-12.

Figure 9. Positive control. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 10. TEFSUC sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 11. SUC sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

Figure 12. WT sample 2 cultivated for 23 hours. Left: overlay channels (bright field, RFP and GFP). Right: RFP channel.

This indicates that the concept of serially connecting promoters could work, but further evaluation could include measuring of fluorescent levels to determine quantitative differences in expression.

Sequence and Features