Part:BBa_K4825020
pAra3
pAra3 is a synthetic inducible promoter designed for automation for yeast, in which the level of the inducer arabinose regulates the initiation of the transcription. AraC functions as a homodimer. In the absence of arabinose, the DNA-binding domain of two subunits binds to Inv AraI1 and AraO2 respectively. Inv represents the inversion of origin element sequence. The two subunits are connected through the dimerisation domain. The 193base pair(bp) intervals and dimerisation enables the DNA loop to form. DNA loop hindered the binding of RNA polymerase. One more copy of AraI and AraO2 are added to this part, thus double DNA loop are expected to be formed to enhance the repression. In the presence of arabinose, two subunits of AraC primarily bind to adjacent Inv Aral1 and Inv Ara2 separated by four bp instead of looping. One more AraC is involved with the same mechanism due to the dublication. The promoter thus becomes accessible, increasing the downstream gene transcription level. In our project, this part is utilized to construct a plasmid with Bl-1 gene and control its transcription by inducing it with the presence of arabinose in S.cerevisiae and prevent the initiation of kill switch.
Usage and Biology
Ara promoter with L-arabinose operon and its downstream araBAD gene regulates the metabolisation of arabinose in E.coli. In natural E.coli cell system, the regulatory protein of Ara operon, AraC, acts positively to stimulate transcription in the presence of arabinose and acts negatively in the absence of arabinose to repress transcription initiation. In the presence of high level of arabinose and low level of glucose, the complex of cAMP and catabolite activator protein(CAP) together with dimeric AraC protein with two subunits binding to AraI 1 and Ara 2 elements respectively, stimulate the initiation. In the absence of arabinose, the dimeric AraC with two subunits binding to AraI1 and AraO2 elements respectively represses the transcription. Two AraI, overall four halfsites and twp AraO2 are selected to design the synthetic Ara3 promoter in this part.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 39
Illegal BamHI site found at 102 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1
- 1000COMPATIBLE WITH RFC[1000]
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