Team:Tartu TUIT/Characterization
One of the criteria for earning a medal is the characterization of biological parts. Besides that, expansion of the open-access Registry of Standard Biological Parts creates more possibilities for future IGEM teams and academic labs to speed up their research. This is why we decided to contribute to the development of the Registry and to characterize new parts.
As the first, we screened the Registry and chose several yeast promoters. Our choice was based on two criteria: 1) promoters are important regulatory elements, and the level and timing of gene expression in many respects depends on the promoter choice; 2) since our project was performed on yeast, we selected yeast promoters because we know how to handle yeast cells and how to performed and troubleshoot different laboratory techniques. Promoters chosen for characterization are listed in Table 1. For promoter characterization, GFP was chosen as a reporter.
Table 1. Promoters characterized.
| Promoter | Type | Gene regulated by the promoter/function | Length/source |
|---|---|---|---|
| pMET25 | Inducible(silent when methionine is present in the growth medium and is activated in methionine drop-out medium) | O-acetyl homoserine-O-acetyl serine sulfhydrylase gene/required for methionine and cysteine biosynthesis | BBa_K165000 |
| pPGK1 | Constitutive | 3-phosphoglycerate kinase/ key enzyme in glycolysis and gluconeogenesis | BBa_K122000 |
| pGAL1 | Inducible (strongly inhibited by glucose and activated by galactose) | Galactokinase/takes part in the metabolism of galactose | 604 bp upstream of the start codon |
| pRNR1 | Constitutive | Major isoform of the large subunit of ribonucleotide-diphosphate reductase/dNTP synthesis | 1000 bp upstream of the start codon |
Methodology
All common methods such as PCR, agarose gel electrophoresis, miniprep, bacterial and yeast transformation, and some others are described in the Experiments section.
Plasmid construction
Target promoters were PCR amplified either from plasmids or from yeast genomic DNA with primers containing SacI (forward primer) and NotI (reverse primer) restriction sites at their 5’-ends. PCR products were separated on the agarose gel, purified and cut with SacI/NotI enzymes. As a backbone, we used pRS306 plasmid containing pTDH3-GFP-tCYC1 cassette. The vector was digested with SacI/NotI restriction enzymes, which cut out the pTDH3 promoter region, and was ligated with our target promoters. After bacterial transformation and miniprep, sequence-verified plasmids were used for yeast transformation.
Yeast strain construction
Constructed vectors with GFP under target promoters were restricted and used for yeast transformation. S. cerevisiae DOM90 (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain was transformed. All the yeast strains generated and used for promoter characterization are listed in Table 2.
Table 2. Yeast strains used for promoter characterization experiments
| Strain name | Genotype* | Description |
|---|---|---|
| DOM0090 | ura3-1::pRS306-URA3 | Strain with empty vector; was used as a negative control for GFP fluorescence |
| TTI_1 | ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 | Strain with EGFP under TDH3 promoter; was used as a positive control for GFP fluorescence |
| TTI_2 | ura3-1::pRS306 pMET25-EGFP-tCYC1-URA3 | Strain with GFP under pMET25 promoter |
| TTI_3 | ura3-1::pRS306-pGAL1-EGFP-tCYC1-URA3 | Strain with GFP under pGAL1 promoter |
| TTI_4 | ura3-1::pRS306 pPGK1-EGFP-tCYC1-URA3 | Strain with GFP under pPGK1 promoter |
| TTI_5 | ura3-1::pRS306 pRNR1-EGFP-tCYC1-URA3 | Strain with GFP under pRNR1 promoter |
*DOM90 strain (MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) was used for transformation. In the Genotype column, only the differences between strains are provided. Following transformation, separate yeast colonies were screened for the presence of the insert under the fluorescent microscope. Colonies displaying GFP fluorescence were selected for further fluorescence measurement experiment.
Fluorescence measurement
Prior to measurements, yeast strains were pregrown in liquid media overnight at 30°C and 200 RPM in the shaker incubator. The next day, optical density (OD600) of yeast cultures was measured. Further procedures slightly differed for the strains carrying constitutive and inducible promoters. Strains with constitutive promoters were diluted with fresh media to reach the final OD600 from 0.8 to 1.1. Proper culture volumes of the strains carrying inducible promoters were first centrifuged, washed with distilled water and resuspended in either inductive or non-inductive media to get final culture OD600 from 0.8 to 1.1. Before the fluorescence measurements, cell cultures were grown for 3 hours at 30°C and 200 RPM in the shaker incubator. All the media used for different strains are listed in Table 3.
Table 3. Media used for cell growth
| Strain | Medium* | Carbon source |
|---|---|---|
| ura3-1::pRS306-URA3 | CSM | 2% glucose |
| ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 | CSM | 2% glucose |
| ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 | CSM | 2% glucose |
| ura3-1::pRS306-pTDH3-EGFP-tCYC1-URA3 | CSM | 2% glucose (non-inductive conditions) 2% galactose (inductive conditions) |
| ura3-1::pRS306 pMET25-EGFP-tCYC1-URA3 | CSM (non-inductive conditions) CSM methionine drop-out (inductive conditions) |
2% glucose |
| ura3-1::pRS306 pPGK1-EGFP-tCYC1-URA3 | CSM | 2% glucose |
After 3 hours of incubation, 200 µL of each strain culture was transferred into a 96-well plate (clear flat bottom). Two biological replicates (two different positive colonies after transformation) of each genotype were taken in four technical replicates for fluorescence measurements. For the fluorescence measurement, we have used BioTek Synergy MX microplate reader with the following setup: excitation 485 nm, emission 528 nm, bandwidth 20, gain 80. Alone with fluorescence, absorbance at 600 nm was measured (with disabled pathlength correction). CSM medium was used as blank for OD600 measurements. Fluorescein was used as a reference (according to the protocol of 2018 iGEM InterLab Study) to quantify GFP fluorescence intensity. However, in our case, the standard curve for fluorescein concentrations in the range from 0.0012 to 0.313 µM was generated, in contrast to the InterLab Study, where 10 µM of fluorescein was the highest concentration. Since we used a higher gain parameter in our experiment, fluorescein concentrations above 0.313 µM showed intensities that were above the detection limit of our device.
Results
For part characterization, three yeast promoters were chosen: constitutive pPGK1, and two inducible pMET25 and pGAL1 (our shorter version of this promoter differs from that available in the Registry). EGFP was cloned under target promoters and constructs were transformed into yeast cells. The intensity of GFP fluorescence was taken as a measure of promoter strength. As can be seen from Figure 1, the highest fluorescence intensity was observed for the constitutive pRNR1 promoter. EGFP under pRNR1 showed 40% higher fluorescence in comparison to a positive control (pTDH3-EGFP). Similar numbers were obtained for induced pMET25 and pGAL1. The lowest fluorescence intensity was observed for EGFP under pPGK1 (72% relative to EGFP fluorescence in the positive control). Surprisingly, EGFP fluorescence was detected in the strains with inducible promoters under non-inductive conditions (presence of methionine for pMET25 promoter and glucose for pGAL1 promoter).
Conclusions
The results of EGFP fluorescence measurements suggest that all three characterized yeast promoters initiate transcription at a similar rate, which was higher in our experiment than one observed for pTDH3 (positive control). Surprisingly, both inducible pGAL1 and pMET25 promoters showed some leakage effect under non-inductive conditions that should be taken into consideration while choosing a promoter for the experiment.