Difference between revisions of "Part:BBa K2100000:Design"

 
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===Design Notes===
 
===Design Notes===
 
Estrogen Side Promoter Design
 
Estrogen Side Promoter Design
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-ERE's are spaced within the locations of the TetO sites of the TRE inducible promoter. Additionally, since the tetO site is 19 base pairs and ERE is only 13 base pairs, we added 5 additional random and different base pairs upstream, directly in front of the ERE sequence, to maintain the geometry of the TRE promoter since the way the DNA folds is important and we didn't want the DNA to misfold by having an incorrect length.  
+
ERE's are spaced within the locations of the TetO sites of the TRE inducible promoter. Additionally, since the tetO site is 19 base pairs and ERE is only 13 base pairs, we added 5 additional random and different base pairs upstream, directly in front of, the ERE sequence to maintain the geometry of the TRE promoter since the way the DNA folds is important and we didn't want the DNA to misfold by having an incorrect length. The ERE sequence has three unidentified base pairs in the middle of its sequence, which we chose to fill randomly and differently for each occurrence of an ERE site since there is no indication that they have any effect on the functionality of the binding to ER-alpha and also because IDT would not synthesize it with so many repeated sequences. We chose repetitions of 3,5, and 6 because the paper Klinge et al. [1] had most success with 3 synthetic EREs, then five was a middle number, and 6 is the number of TetO sites on TRE-tight that we wanted to mimic since that was a successful synthetically built inducible promoter.  
−
-ERE sequence has three blank base pairs in the middle of its sequence, which we chose to fill randomly and differently for each occurence of an ERE site since there is no indication that they have any effect on the functionality of the binding to ER-alpha and also because IDT would not synthesize it with so many repeated sequences.
+
We additionally liked the fact that the synthetic pEREs drastically shortened the length of promoter from the 2000+ base pairs of the natural estrogen responsive promoter, pTFF1. We also decided to try to synthesize an inducible promoter rather than doing the fusion with VP16-GAL4 since we wanted to maintain and utilize as many natural components of the cell itself.
−
-Chose repetitions of 3,5, and 6 because the paper had most success with 3 synthetic EREs, then five was a middle number, and 6 is the number of TetO sites on TRE-tight that we wanted to mimic since that was a successful synthetically built inducible promoter.  
+
[1]
−
-Choose pTFF1 because that is a natural promoter that responds to ER-alpha.
+
−
-We additionally liked the fact that the synthetic pEREs drastically shortened the length of promoter from the 2000+ base pairs of pTFF1.
+
−
-We also decided to try to synthesize an inducible promoter rather than doing the fusion with VP16-GAL4 (like the progesterone side) since we wanted to maintain and utilize as many natural components of the cell itself.
+
−
 
+
  
  

Revision as of 22:18, 17 October 2016


pENTR pEREx3


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 180
    Illegal EcoRI site found at 190
  • 21
    INCOMPATIBLE WITH RFC[21]
    Unknown
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 180
    Illegal EcoRI site found at 190
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 6
    Illegal EcoRI site found at 180
    Illegal EcoRI site found at 190
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Estrogen Side Promoter Design ERE's are spaced within the locations of the TetO sites of the TRE inducible promoter. Additionally, since the tetO site is 19 base pairs and ERE is only 13 base pairs, we added 5 additional random and different base pairs upstream, directly in front of, the ERE sequence to maintain the geometry of the TRE promoter since the way the DNA folds is important and we didn't want the DNA to misfold by having an incorrect length. The ERE sequence has three unidentified base pairs in the middle of its sequence, which we chose to fill randomly and differently for each occurrence of an ERE site since there is no indication that they have any effect on the functionality of the binding to ER-alpha and also because IDT would not synthesize it with so many repeated sequences. We chose repetitions of 3,5, and 6 because the paper Klinge et al. [1] had most success with 3 synthetic EREs, then five was a middle number, and 6 is the number of TetO sites on TRE-tight that we wanted to mimic since that was a successful synthetically built inducible promoter. We additionally liked the fact that the synthetic pEREs drastically shortened the length of promoter from the 2000+ base pairs of the natural estrogen responsive promoter, pTFF1. We also decided to try to synthesize an inducible promoter rather than doing the fusion with VP16-GAL4 since we wanted to maintain and utilize as many natural components of the cell itself. [1]


Source

DNA sequence synthesized by IDT.

References