Difference between revisions of "Part:BBa K165000"
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INTRODUCTION | INTRODUCTION | ||
| − | <p>In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed | + | <p>In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under MET25 promoter. The empty plasmid was used as a negative control. |
</p><p>MATERIALS AND METHODS | </p><p>MATERIALS AND METHODS | ||
Revision as of 12:43, 16 October 2019
MET 25 Promoter
The is a repressible promoter for use in Saccharomyces cerevisiae. It is repressed by the presence of methionine. When unrepressed, is has a moderate level of activity.
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]
INTRODUCTION
In order to determine the strength of pMET25 promoter, we have conducted the following experiment. First of all, we have constructed a plasmid where EGFP is expressed under MET25 promoter. 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 pMET25-EGFP-tCYC1 | MET25 | EGFP | tCYC1 | pRS306 |
| 2 | 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 |
| Met25 uninduced | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 pMET25-EGFP-tCYC1 |
| Met25 induced | DOM90 | MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] | pRS306 pMET25-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 CSM/2%Glc media, resuspended in fresh CSM/2%Glc media to OD600 ca. 0.7, divided into 2 parts: half was induced by changing media from CSM to -Met, half was left uninduced. Put to grow for 3 hours. 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 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 | Met25 uninduced | MET25 induced | SCM | |
| Colony 1 Replicate 1 | ||||
| Colony 1 Replicate 2 | ||||
| Colony 1 Replicate 3 | ||||
| Colony 1 Replicate 4 | ||||
| Colony 1 Replicate 5 | ||||
| Colony 1 Replicate 6 | ||||
| Colony 1 Replicate 7 | ||||
| Colony 1 Replicate 8 |
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