Difference between revisions of "Part:BBa K3019017"
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|pRS306 pTDH3-EGFP-tCYC1 | |pRS306 pTDH3-EGFP-tCYC1 | ||
| − | |TDH3 | + | |TDH3 |
| + | |EGFP | ||
| + | |tCYC1 | ||
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Revision as of 21:53, 15 October 2019
pGAL promoter from S. cerevisiae
Galactose inducible promoter of S. cerevisiae -804 to 0 from GAL1 start codon.
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 421
- 1000COMPATIBLE WITH RFC[1000]
INTRODUCTION
In order to determine the strength of pGAL1 promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. As a positive control we used pTDH3. 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 pGAL1-EGFP-tCYC1 | GAL1 | EGFP | tCYC1 | pRS306 |
| 2 | pRS306 pTDH3-EGFP-tCYC1 | TDH3 | EGFP | tCYC1 | |
| 3 | pRS306 | ||||
After construction of plasmids was finished, we have transformed S. cerevisiae DOM90 (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain with plasmids listed above to create the strains (table 2).
Table 2. S. cerevisiae strains created.
| Strain name | Genotype | Plasmid integrated | |
| Negative control | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 |
| Positive control(TDH3) | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 pTDH3-EGFP-tCYC1 |
| pGAL1+glucose | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 pGAL1-EGFP-tCYC1 |
| pGAL+galactose | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 pGAL1-EGFP-tCYC1 |
We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
pGAL+glucose was pregrown overnight at 30°C in liquid CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 ca. 0.7, pGAL+galactose was pregrown overnight at 30°C in liquid CSM/2%Gal media, resuspended in fresh CSM/2%Gal media to OD600 ca. 0.7 Distributed to 96 well plate (clear flat bottom). 8 replicates from the strain were used. 200 µL of cells were added to each well. As a reference to OD600, fresh CSM/2%Glc or CSM/2%Gal 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 | pGAL+glucose | pGAL+galactose | |
| Colony 1 Replicate 1 | ||||
| Colony 1 Replicate 2 | ||||
| Colony 1 Replicate 3 | ||||
| Colony 1 Replicate 4 | ||||
| Colony 2 Replicate 1 | ||||
| Colony 2 Replicate 2 | ||||
| Colony 2 Replicate 3 | ||||
| Colony 2 Replicate 4 |
As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according to 2018 iGEM InterLab Study. 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.
Conclusions: