Difference between revisions of "Part:BBa K2110012"
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<partinfo>BBa_K2110012 short</partinfo>
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<h1>Contribution</h1>
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This promoter is a enhanced promoter used in Saccharomyces cerevisiae. It regulates the 3-phosphoglycerate kinase (PGK1) gene in Saccharomyces cerevisiae. This promoter can be used in both plasmid and genome of Saccharomyces cerevisiae.
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===Usage and Biology===
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<p>During our project we had a great deal of work with different types of promoters - constitutive and indusible. During the planning of the project and the accumulation of knowledge about the various parts, we discovered that much information is lacking about many of these parameters, and the information that exists exists scattered among many places and is not arranged in one place. We chose two modulators - TDH3 [<a href="BBa_K124002"target="_blank">https://parts.igem.org/Part:BBa_K124002</a>] of team iGEM08_Brown and PGK1 [<a href="https://parts.igem.org/Part:BBa_K2110012" target="_blank">https://parts.igem.org/Part:BBa_K2110012</a>]of team  iGEM16_Tianjin, two strong constitutive permutative promoters, and improved their descriptions and information about them, referring to the main sources we used. We hope that groups coming after us will have faster and more convenient access to information when they come to choose the parameters they want to use.</p>
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===Sequence and Features===
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<h2>Improve an existing part</h2>
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<partinfo>BBa_K2110012 SequenceAndFeatures</partinfo>
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<p>We chose to take the existing BBA part of miraculin, and improve it by adding the HXT7 promoter we have already isolated.</p>
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<p>Our new part is HXT7_miraculin - BBa_K2408024 <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2408024" target="_blank">https://parts.igem.org/wiki/index.php?title=Part:BBa_K2408024</a></p>
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Group: Tel-Hai 2017
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<p>HXT7 transcription was repressed at high glucose levels and was detected when the glucose in the culture approached depletion.</p>
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<p>All hexose transporter proteins Hxt2, 4, 6 and 7 in S. cerevisiae are repressed by high glucose concentration, and induced when glucose concentration decreases below a certain level.</p>
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Author: Yaakov Bulka
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<p>HXT7 seems to bind glucose with the highest affinity among all glucose transporters, and this fact is associated to a strong induction at low glucose level. The HXT7 promoter region turned out to be suitable for recombinant protein production in yeast and was compared to other yeast promoters (PTEF1, PADH1, PTPI1, PPGK1, PTDH3 and PPYK1) using lacZ as a reporter gene. Among them, PHXT7 was stated as the strongest promoter in continuous culture with limited glucose level.</p>
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Summary: we added information about the promoter and on its mechanism.
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<a href="https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-13-5#CR44" target="_blank">https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-13-5#CR44</a>
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<a href="http://onlinelibrary.wiley.com/doi/10.1002/yea.771/pdf" target="_blank">http://onlinelibrary.wiley.com/doi/10.1002/yea.771/pdf</a>
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PGK1 is the promoter of the gene for phosphoglycerate kinase, a key glycolytic gene. The protein is involved in step 2 of the subpathway that synthesizes pyruvate from D-glyceraldehyde 3-phosphate. In the literature it is generally considered a strong promoter. PGK1 promoter shows very constant activity pattern at different glucose concentrations.
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</p>
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Studies have found that The PGK gene has an upstream activation sequence located between 402 and 479 nucleotides upstream from the initiating ATG sequence, which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation.
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<p>Since transcription of genes with the HXT7 promoter was correlated with the extracellular glucose concentration in the cultures, we can cause the miraculin to be released only when the glucose levels are low and thus adjust the level of its secretion to the desired taste in wine - at a high concentration of glucose it will not be dispensed, and at low concentration it will be secreted and improve the wine's sweet taste.</p>
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The activation sequence between bases -538 and -402 upstream of the initiating ATG contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. those studies have also found that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1.  
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Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence.  
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Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955-964.‏
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Ogden, J. E., Stanway, C., Kim, S., Mellor, J., Kingsman, A. J., & Kingsman, S. M. (1986). Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences. Molecular and cellular biology, 6(12), 4335-4343.‏
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Chambers, A., Tsang, J. S., Stanway, C., Kingsman, A. J., & Kingsman, S. M. (1989). Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1. Molecular and cellular biology, 9(12), 5516-5524.‏
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===Contribution: NUDT_CHINA 2019===
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Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by the expression of firefly luciferase reporter gene in HepG2 cells.  
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==Methods==
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To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying PGK promoter-firefly luciferase cassette was transfected into liver carcinoma cell line HepG2 by lipo3000 under standard proytocol. Cells were cultures under standard DMEM High glucose medium under 5% CO2 and 37 ℃. Cells were harvested 0, 6, 12 and 24h after transfection, and firefly luciferase activity was measured by Beyotime™  Dual Luciferase Reporter Gene Assay Kit.
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==Results==
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The result showed a significant increase of firefly luciferase within 24 hours of transfection.
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https://static.igem.org/mediawiki/parts/thumb/f/fd/T--NUDT_CHINA--2019_Firefly.png/303px-T--NUDT_CHINA--2019_Firefly.png
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Figure 1. PGK-drived firefly luciferase activity, Error bar represents SD of at least 3 biological replicates.
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2110012 parameters</partinfo>
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Latest revision as of 02:25, 22 October 2019


PGK1 promoter

This promoter is a enhanced promoter used in Saccharomyces cerevisiae. It regulates the 3-phosphoglycerate kinase (PGK1) gene in Saccharomyces cerevisiae. This promoter can be used in both plasmid and genome of Saccharomyces cerevisiae.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Group: Tel-Hai 2017

Author: Yaakov Bulka

Summary: we added information about the promoter and on its mechanism.


PGK1 is the promoter of the gene for phosphoglycerate kinase, a key glycolytic gene. The protein is involved in step 2 of the subpathway that synthesizes pyruvate from D-glyceraldehyde 3-phosphate. In the literature it is generally considered a strong promoter. PGK1 promoter shows very constant activity pattern at different glucose concentrations. Studies have found that The PGK gene has an upstream activation sequence located between 402 and 479 nucleotides upstream from the initiating ATG sequence, which is required for full transcriptional activity. Deletion of this sequence caused a marked reduction in the levels of PGK transcription. PGK has no requirement for TATA sequences; deletion of one or both potential TATA sequences had no effect on either the levels of PGK expression or the accuracy of transcription initiation. The activation sequence between bases -538 and -402 upstream of the initiating ATG contains multiple functional elements, including an essential region called the activator core (AC) sequence and three copies of the pentamer 5'-CTTCC-3'. The AC sequence shows strong homology to the consensus binding sites for the yeast proteins RAP1 (GRF1) and TUF. those studies have also found that the yeast protein which interacts with the AC sequence is the DNA-binding protein RAP1. Expression of the PGK gene is found to be regulated according to the carbon source in the growth medium. PGK mRNA levels are high in yeast cells grown in glucose medium but low in yeast cells grown in media containing carbon sources such as pyruvate and acetate. This carbon source regulation of transcription was found to be mediated, in part, via regulation of RAP1 binding to the AC sequence.


Partow, S., Siewers, V., Bjørn, S., Nielsen, J., & Maury, J. (2010). Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast, 27(11), 955-964.‏

Ogden, J. E., Stanway, C., Kim, S., Mellor, J., Kingsman, A. J., & Kingsman, S. M. (1986). Efficient expression of the Saccharomyces cerevisiae PGK gene depends on an upstream activation sequence but does not require TATA sequences. Molecular and cellular biology, 6(12), 4335-4343.‏

Chambers, A., Tsang, J. S., Stanway, C., Kingsman, A. J., & Kingsman, S. M. (1989). Transcriptional control of the Saccharomyces cerevisiae PGK gene by RAP1. Molecular and cellular biology, 9(12), 5516-5524.‏


Contribution: NUDT_CHINA 2019

Summary：in this part, we made contributions to the quantitative characterization data of mammalian PGK Promoter. We measured the strength of this promoter by the expression of firefly luciferase reporter gene in HepG2 cells.

Methods

To characterize the PGK promoter, firefly luciferase was used as the reporter gene to quantify the transcriptional strength of promoter. To be specific, plasmids carrying PGK promoter-firefly luciferase cassette was transfected into liver carcinoma cell line HepG2 by lipo3000 under standard proytocol. Cells were cultures under standard DMEM High glucose medium under 5% CO2 and 37 ℃. Cells were harvested 0, 6, 12 and 24h after transfection, and firefly luciferase activity was measured by Beyotime™ Dual Luciferase Reporter Gene Assay Kit.

Results

The result showed a significant increase of firefly luciferase within 24 hours of transfection.

303px-T--NUDT_CHINA--2019_Firefly.png


Figure 1. PGK-drived firefly luciferase activity, Error bar represents SD of at least 3 biological replicates.