Part:BBa_K2100027

pEXPR pERE3:eYFP

This construct is an expression vector containing the pERE3 promoter upstream of a yellow fluorescent protein (eYFP). pEREx3 is a synthetic mammalian promoter that responds to estrogen receptor activated by estradiol (E2). pEREx3 consists of three repeats of the estrogen response element (ERE) consensus sequence upstream of a minimal promoter (minCMV).

In endometrial cells, estrogen receptors facilitate the cell's response to estrogen, initiating the proliferative phase of the menstrual cycle. Mechanistically, estrogen diffuses through the cell membrane and binds to the ligand binding domain of the ER. The DNA-binding domain of the estrogen receptor becomes exposed. The receptor then relocalizes to the nucleus from the cytoplasm, recruits co-activators, and acts as a transcription factor. The eYFP gene is then transcribed. There are two isoforms of the estrogen receptor, ER alpha and ER beta. The two differ in their ligand selectivity. In the context of our project, we are working with ER alpha.

Our team tested different possible variations of an estrogen-responsive promoter by varying the amount of estrogen response elements (EREs) present for the activated ER complex to bind to. This particular construct contains three estrogen responsive elements with the sequence 5′-GGTCAnnnTGACC-3′. We interspaced these elements with 22 randomly selected bases in between, the same spacing between TREs in the TRE promoter. As noted from the results of the paper Klinge et. al. 1997[1], increasing the number of EREs does not correlate with an increase in transcriptional activity, possibly due to helical turns in the DNA that change the orientation of an ERE depending on the number and spacing of them. When an ERE is not properly oriented, it has trouble interacting with ERα and polymerases. Since we wanted to decrease the probability of helical turns inhibiting the transcriptional activity of promoters, we modeled the spacing after the TRE promoter.

[1]

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 180
  Illegal EcoRI site found at 190
  Illegal PstI site found at 428
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 180
  Illegal EcoRI site found at 190
  Illegal PstI site found at 428
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 180
  Illegal EcoRI site found at 190
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 180
  Illegal EcoRI site found at 190
  Illegal PstI site found at 428
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 6
  Illegal EcoRI site found at 180
  Illegal EcoRI site found at 190
  Illegal PstI site found at 428
  Illegal AgeI site found at 211
  
• 1000
  COMPATIBLE WITH RFC[1000]