Part:BBa_K431008
pTEF ( Translation elongation factor promoter)
pTEF The TEF1- α gene is comprised of 1.380 kB and encodes a polypeptide of 459 amino acids and a molecular weight of 50.1 kDa. The final product is the soluble protein Translational Elongation Factor. This protein has been determined to be one of the most abundant proteins in eukaryotic cells and known to be associated with cell growth. Recognizing the high level of the protein present in the cell, it was predicted that TEF1- α is under the control of a strong promoter. It was discovered that the translational elongation factor is under the control of strong constitutive promoter pTEF.pTEF was then isolated and compared with another strong constitutive promoter, glyceraldehydes-3-phosphate dehydrogenase promoter (pGAP). According to research done on this promoter it has been determine that when compared to pGAP, pTEF was shown to work well in high glucose environments and carbon-limited conditions, therefore leading to similar or even greater expression levels of recombinant protein. It was also determined that pTEF does not require an inducer, since it is a constitutive promoter. pTEF can also be used in the mass production of heterologuous proteins. From the research done on this promoter it was concluded that pTEF can provide more choices on production conditions and easier methods for expression than pGAP. Therefore, pTEF would be a great alternative for expressing foreign genes in Pichia pastoris for industrial application. This promoter sequence can be isolated from yeast Pichia pastoris. The promoter sequence was also scanned for the restriction enzymes in all assembly standards to determine if it could be use for iGEM and it was determined to be compatible with all the standards.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
ITRODUCTION
In order to determine the strength of pTEF1 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. We have chosen pTDH3 as a positive control due to its wide use in yeast research. The empty plasmid was used as a negative control.
MATERIALS AND METHODS
Plasmids used to conduct the experiment are listed in table 1.
Table 1. Plasmids created
| Insert | |||||
| number | Plasmid name | Promoter | Gene | Terminator | backbone |
| 1 | pRS306 pTDH3-EGFP-tCYC1 | TDH3 | EGFP | tCYC1 | pRS306 |
| 2 | pRS306 pTEF1-EGFP-tCYC1 | TEF1 | EGFP | tCYC1 | pRS306 |
After construction of plasmids was finished, we have transformed S. cerevisiae DOM90 (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG) strain with plasmids listed above to create the strains (table 2).
Table 2. S. cerevisiae strains created.
| Strain name | Genotype | Plasmid integrated | |
| Positive control | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 |
| Negative control | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 |
| Test | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 |
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
All strains were pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 in the range of 0.1 to 0.9 and distributed to 96 well plate (clear flat bottom). 2 replicates of 4 colonies from each strain were used. 200 µL of cells were added to each well. As a reference to OD600, fresh CSM/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.
Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.
| Negative control | Positive control | pCYC16 | |
| Colony 1 Replicate 1 | |||
| Colony 1 Replicate 2 | |||
| Colony 2 Replicate 1 | |||
| Colony 2 Replicate 2 | |||
| Colony 3 Replicate 1 | |||
| Colony 3 Replicate 2 | |||
| Colony 4 Replicate 1 | |||
| Colony 4 Replicate 2 |
| None |