Difference between revisions of "Part:BBa K1652000"
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With the GEV protein, pPGK1Gx exhibits a U-shaped dose response. GEV is a fusion protein consisting of Gal4 binding protein, human Estrogen receptor, and VP16 (a domain that recruits RNA polymerase). This promoter was created to re-purpose GEV, which is usually an activator, into a repressor. GEV activity is modulated by adding beta-estradiol to the culture. Beta-estradiol up-regulates GEV, so a higher drug concentration should increase repression. Indeed, at low concentrations of the drug, repression is seen. However, at very high concentrations, the VP16 domain on GEV actually recruits polymerase and ''activates'' the gene, although the expression level of the promoter does not reach basal levels even with an extremely high dose.
 
With the GEV protein, pPGK1Gx exhibits a U-shaped dose response. GEV is a fusion protein consisting of Gal4 binding protein, human Estrogen receptor, and VP16 (a domain that recruits RNA polymerase). This promoter was created to re-purpose GEV, which is usually an activator, into a repressor. GEV activity is modulated by adding beta-estradiol to the culture. Beta-estradiol up-regulates GEV, so a higher drug concentration should increase repression. Indeed, at low concentrations of the drug, repression is seen. However, at very high concentrations, the VP16 domain on GEV actually recruits polymerase and ''activates'' the gene, although the expression level of the promoter does not reach basal levels even with an extremely high dose.
  
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'''Dose response of pPGK1Gx to beta-estradiol'''
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=== Improvement ===
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This part is an improvement to part [https://parts.igem.org/Part:BBa_K1400001 K1400001] from uOttawa's 2014 iGEM project. This part, pTREGx, is a promoter that is induced by rtTA but repressed with GEV. We kept the GEV-repressibility, but made it so that the promoter has strong basal expression. This way, the gene does not need a high concentration of GEV in order to get a strong signal.
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Read more on uOttawa's [http://2015.igem.org/Team:uOttawa/Description wiki].
 +
 
 +
=== Dose response of pPGK1Gx to beta-estradiol ===
 
<html><img src="https://static.igem.org/mediawiki/2015/8/8a/Uo2015-results.jpg"></html>
 
<html><img src="https://static.igem.org/mediawiki/2015/8/8a/Uo2015-results.jpg"></html>
  

Revision as of 01:46, 19 September 2015

pPGK1Gx

pPGK1Gx is a constitutive promoter that is able to be repressed by Gal4 transcription factor and its derivatives.

With the GEV protein, pPGK1Gx exhibits a U-shaped dose response. GEV is a fusion protein consisting of Gal4 binding protein, human Estrogen receptor, and VP16 (a domain that recruits RNA polymerase). This promoter was created to re-purpose GEV, which is usually an activator, into a repressor. GEV activity is modulated by adding beta-estradiol to the culture. Beta-estradiol up-regulates GEV, so a higher drug concentration should increase repression. Indeed, at low concentrations of the drug, repression is seen. However, at very high concentrations, the VP16 domain on GEV actually recruits polymerase and activates the gene, although the expression level of the promoter does not reach basal levels even with an extremely high dose.

Improvement

This part is an improvement to part K1400001 from uOttawa's 2014 iGEM project. This part, pTREGx, is a promoter that is induced by rtTA but repressed with GEV. We kept the GEV-repressibility, but made it so that the promoter has strong basal expression. This way, the gene does not need a high concentration of GEV in order to get a strong signal.

Read more on uOttawa's [http://2015.igem.org/Team:uOttawa/Description wiki].

Dose response of pPGK1Gx to beta-estradiol

This figure shows the response of pPGK1Gx driving GFP. Concentration of beta-estradiol is given on the x-axis in nM. Fluorescence is given in arbitrary units on the y-axis. The two data series represent two biological replicates.

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]