Difference between revisions of "Part:BBa K4825019"

 
 
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pAra2 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. 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. 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.  
 
pAra2 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. 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. 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.  
  
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===Usage and Biology===
 
===Usage and Biology===
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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 half sites and AraO2 are selected to design the synthetic Ara2 promoter in this part.
  
 
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Latest revision as of 13:05, 12 October 2023


pAra2

pAra2 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. 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. 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 half sites and AraO2 are selected to design the synthetic Ara2 promoter in this part.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 46
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1
  • 1000
    COMPATIBLE WITH RFC[1000]