Introduction
In order to determine the strength of pRPL18B 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.
MATERIAL AND METHOD
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 pRPL18B-EGFP-tCYC1 | pRPL18B | EGFP | tCYC1 | pRS306 |
| 3 | pRS306 | - | - | - | pRS306 |
After construction of plasmids was finished, we have transformed S. cerevisiae CEN.PK-2-1C strain with plasmids listed above to create the strains (table 2).
Table 2. S. cerevisiae strains created.
| Strain name | Genotype | Plasmid integrated | |
| positive control | YST1 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 pTDH3-EGFP-tCYC1 |
| Negative control | YST2 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 | Test | YST3 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 pRPL18B-EGFP-tCYC1 |
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader. All strains were pregrown overnight at 30°C in liquid –URA/2%Glc media, resuspended in fresh -URA/2%Glc media to OD600 ca. 1 and distributed to 96 well plate (clear flat bottom). 3 replicates from each strain were used. 100 µL of cells were added to each well. As a reference to OD600, fresh -URA/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.
| pRPL18B | ||||||||||
| Negative control | Positive control | Colony 1 | Colony 2 | Colony 3 | Colony 4 | Colony 5 | Colony 6 | -Ura/2% Glc | ||
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||
| Replicate 1 | A | |||||||||
| Replicate 2 | B | |||||||||
| Replicate 3 | C | |||||||||
As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according with 2018 iGEM InterLab Study [2]. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements. The layout of the 96 well plates is given in table 4.
Table 4. The layout of the plate for Fluorescein standard curve measurements.
| Fluorescein µM | 0,313 | 0,157 | 0,078 | 0,039 | 0,0196 | 0,0098 | 0,0049 | 0,0024 | 0 | |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | ||
| Replicate 1 | A | 79055 | 39438 | 20511 | 10034 | 5020 | 2731 | 1302 | 745 | 78 |
| Replicate 2 | B | 80361 | 40466 | 20735 | 10572 | 5367 | 2751 | 1371 | 784 | 86 |
| Replicate 3 | C | 76390 | 38680 | 19692 | 10112 | 5078 | 2719 | 1462 | 866 | 111 |
| Replicate 4 | D | 77470 | 38863 | 19833 | 10170 | 5235 | 2773 | 1344 | 765 | 121 |
Results
The results are presented in figure 1. As it turned out, the pRPL18B promoter is weaker than pTDH3 but still active. This corresponds to the data from the source article [1].
Figure 1
CONCLUSION
We think the characterisation of the promoters is necessary to provide researchers with useful information and toolbox of promoters of different strength to better optimise synthetic pathways.
- Denby, C. M., Li, R. A., Vu, V. T., Costello, Z., Lin, W., Chan, L. J. G., ... & Scheller, H. V. (2018). Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer. Nature communications, 9(1), 965.
- https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf, calibration 3