Part:BBa_K2100052
pEXPR pERE6:TP901
This is a cascade of our synthetic promoter, pERE6, with a serine recombinase, TP901.
pEREx6 is a synthetic mammalian promoter that responds to estrogen receptor activated by estradiol (E2). pEREx6 consists of six 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. 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 six 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, 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.
TP901 is a serine integrase or serine recombinase, which can invert sequences. Some recombinases exhibit unidirectionality, meaning that once they reverse or cut out the sequence, this action cannot be undone. This means that instead of behaving like a switch, capable of turning on or off, unidirectional recombinases behave as latches.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal EcoRI site found at 773
Illegal EcoRI site found at 1876
Illegal XbaI site found at 42 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal EcoRI site found at 773
Illegal EcoRI site found at 1876 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal EcoRI site found at 773
Illegal EcoRI site found at 1876
Illegal BglII site found at 1656
Illegal BamHI site found at 360 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal EcoRI site found at 773
Illegal EcoRI site found at 1876
Illegal XbaI site found at 42 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 6
Illegal EcoRI site found at 18
Illegal EcoRI site found at 333
Illegal EcoRI site found at 343
Illegal EcoRI site found at 773
Illegal EcoRI site found at 1876
Illegal XbaI site found at 42
Illegal NgoMIV site found at 524 - 1000COMPATIBLE WITH RFC[1000]
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