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===Usage and Biology=== !-->
==Estonia_TUIT2020 Contribution==

==Promoter characterization based on research articles==
The main features of the promoters chosen for characterization are listed in Table 1.

{| class="wikitable" style="margin: auto;"
|+Table 1. Characterized promoter parts.
|-
!Promoter
!Type
!Gene regulated by the promoter/function
|-
|STE5
|Induced by α-factor-pheromone (Printen & Sprague, 1994)
|Ste5 scaffold protein; ensures assembly of the protein kinases of the pheromone-activated MAPK cascade into an active complex during mating (Printen & Sprague, 1994)
|}

STE5 promoter controls the transcription of the STE5 gene, which encodes for the Ste5 scaffold protein. The scaffold protein promotes the assembly of a big protein complex, which will induce cell-cycle arrest and mating (Printen & Sprague, 1994).
The comparative study on the abundance of the Ste5 scaffold protein depending on the promoter was conducted by Chapman & Asthagiri in 2009 ('''Figure 1'''). As can be seen from Figure 1, the STE5 promoter is a very weak inducible promoter, even in comparison with the constitutive CYC promoter, which ensures a very low level of transcription of the regulated gene
[[File:1(2).jpg|thumb|center|500px|Figure 1.]] The relative abundance of STE5-MYC-tagged protein expressed from inducible STE5 (induced by α-factor) and constitutive CYC, ADH, TEF, and GPD promoters. The error bars represent SEM (n=3) (Chapman & Asthagiri, 2009).

==Methology==
PCR, agarose gel electrophoresis, plasmid extraction, bacterial, and yeast transformations, are described in the Estonia_TUIT2020 Materials and Methods section.

===Plasmid construction===
Target promoters were PCR amplified 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 restricted with SacI/NotI according to the instruction manual. As a backbone, we used the pRS306 plasmid vector containing the pTDH3-EGFP-tCYC1 cassette. The vector was SacI/NotI digested to cut out the pTDH3 promoter. Products were separated on the gel and the band of the expected size was purified. After ligation with STE5 promoters, bacterial transformation, and miniprep, sequence-verified plasmids were used for yeast transformation.

===Yeast strain construction===
Constructed integration vectors with EGFP under target promoters were restricted with NcoI 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. Transformants were selected for URA+ phenotype on uracil dropout CSM plates containing 2% glucose. All the yeast strains generated and used for promoter characterization are listed in Table 2.

{| class="wikitable" style="margin: auto;"
|+Table 2. Yeast strains used in promoter characterization experiments
|-
!Strain name
!Genotype*
!Description
|-
|DOM0090
|MATa {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]
|Control strain; was used as a background control for EGFP fluorescence
|-
|ET43
|ura3-1::pRS306-pTDH3-EGFP-tCYC1
|Strain with EGFP under TDH3 promoter; was used as a positive control for EGFP fluorescence (this study)
|-
|ET45
|ura3-1::pRS306-pSTE5-EGFP-tCYC1
|Strain with EGFP under STE5 promoter (this study)
|}

*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 indicated. Following transformation, single yeast colonies were screened for the presence of the insert under the fluorescent microscope. Colonies displaying EGFP fluorescence were selected for further experiments.

===Time-lapse microscopy===
Before time-lapse microscopy, the yeast strain cultures were grown in either synthetic complete (CSM; DOM0090 strain) or in CSM-uracil dropout (CSM-URA; ET43, ET44, ET45 strains) media. The cultures were grown to OD600 0.2-0.8. After that, 0.5 µl of each strain culture was pipetted onto a 0.08 mm cover glass slip and covered with 1.5% agar-SCM (low melting point agarose was used) with or without α-factor at a final concentration of 1 µg/ml  (corresponds to approx. 600 nM). Zeiss Observer Z1 microscope with an automated stage, 63C/1.4NA oil immersion objective, and Axiocam 506 mono camera was used for imaging. During time-lapse imagining the focus was kept using Definite Focus and the sample was kept at 30 °C using PeCon TempControl 37-2 digital. The cells were imaged every 3 minutes and the experiments were 5 hours long. In total, cell images from 10 positions were taken using ZEISS ZEN software. At every time point, EGFP expressing cells were exposed for 15 ms using a Colibri 470 LED module (used at 25% power). MATLAB (The MathWorks, Inc.) was used for image segmentation, cell tracking, and quantification of the levels of fluorescent signals as described in Doncic et al. (2013). For every strain, data represents the average fluorescence from all the cells after 4 hours of imaging. 

==Results==
To evaluate the strength of the STE5 promoter, EGFP was cloned under target promoters and constructs were integrated into the yeast genome. The intensity of EGFP fluorescence in the constructed strains was analyzed by quantification of the fluorescent signals from single cells of time-lapse microscopy images (Figure 4). The intensity of EGFP fluorescence was taken as a measure of promoter strength. DOM0090 was used as a background control for EGFP fluorescence. ET43 strain, which carries EGFP under a strong constitutive TDH3 promoter was used as a positive control.

[[File:T--Estonia TUIT--contr-fig4.jpg|thumb|center|500px|Figure4.]]

The quantification of the fluorescent signals showed that STE5 is a very weak α-factor-inducible promoters (Figure 5). After induction, promoter ensured weak expression level, which is, however, distinguishable from the background, and much lower from those for TDH3 promoter (approx. 9 times lower for STE5 promoter). Under conditions tested, the EGFP signal expressed from STE2 was twice higher in comparison to the STE5 promoter.

[[File:T--Estonia TUIT--contr-fig5.jpg|thumb|center|500px|Figure5.]]

Our results are in agreement with the literature data on the low abundance of the protein expressed from the Ste5 promoter (Fig. 1).

==Conclusions==
We analyzed the strength of two α-factor-inducible yeast promoters STE2 and STE5. The results indicate that the promoters can be used in the experiments when low gene expression levels are required and α-factor can be used for promoter induction. However, a possible promoter leakage in the absence of an activation signal needs to be considered.  

===References===
• Chapman, S. A., & Asthagiri, A. R. (2009). Quantitative effect of scaffold abundance on signal propagation. Molecular Systems Biology, 5(1), 313. https://doi.org/10.1038/msb.2009.73
Di Segni, G., Gastaldi, S., Zamboni, M., & Tocchini-Valentini, G. P. (2011). Yeast pheromone receptor genes STE2 and STE3 are differently regulated at the transcription and polyadenylation level. Proceedings of the National Academy of Sciences of the United States of America, 108(41), 17082–17086. https://doi.org/10.1073/pnas.1114648108

• Doncic, A., Eser, U., Atay, O., & Skotheim, J.M. (2013). An algorithm to automate yeast segmentation and tracking. PLoS One, 8, p. e57970. https://doi.org/10.1371/journal.pone.0057970

• Hartig, A., Holly, J., Saari, G., & Mackayl2, V. L. (1986). Multiple Regulation of STE2, a Mating-Type-Specific Gene of Saccharomyces cerevisiae. In MOLECULAR AND CELLULAR BIOLOGY (Vol. 6, Issue 6). http://mcb.asm.org/

• Jenness, D. D., Burkholder, A. C., & Hartwell, L. H. (1986). Binding of alpha-factor pheromone to Saccharomyces cerevisiae a cells: dissociation constant and number of binding sites. Molecular and Cellular Biology, 6(1), 318–320. https://doi.org/10.1128/mcb.6.1.318

• Printen, J. A., & Sprague, G. F. (1994). Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics, 138(3), 609–619.

• Youk, H., & Lim, W. A. (2014). Secreting and sensing the same molecule allows cells to achieve versatile social behaviors. Science, 343(6171). https://doi.org/10.1126/science.1242782

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_I766557 SequenceAndFeatures</partinfo>
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