Composite
Part:BBa_K3519010
Designed by: Omkar Mohapatra Group: iGEM20_IISER-Tirupati_India (2020-10-07)
Revision as of 11:45, 24 October 2020 by Omkarmohapatra123 (Talk | contribs)
araC-araBAD promoter
This part contains the araC gene downstream to a medium expression system in the opposite direction. The araBAD promoter is integrated downstream to this system.
Usage and Biology
The araC protein is constitutively produced by the medium strength Anderson promoter (J23118) and the strong RBS (B0034) and production of any downstream gene (gene of interest) is controlled by the P-araBAD.
The AraC protein forms a dimer, each molecule binds to the araI1 and araO2 regulatory regions of the DNA and prevents the binding of RNA polymerase to the P-araBAD promoter region and hence represses the transcription of the downstream gene.
In the presence of Arabinose (and low amount of glucose sugar), the Arabinose binds to the AraC protein dimer, brings about a conformational change and binds to the regions araI1 and araI2 regions of the DNA releasing the P-araBAD promoter. Now, RNA polymerase binds to the P-araBAD promoter and proceeds with transcription. The incorporation of RNA polymerase at the P-araBAD promoter is enhanced by the CAP-cAMP complex bound to the CAP binding site. As the concentration of cAMP is inversely proportional to the concentration of glucose, for a constant amount of CAP protein, lesser the concentration of glucose, more will be the concentration of cAMP molecules, hence enhanced transcription of any downstream gene.
Figure 1: The designed arabinose inducible promoter system. The araC is constitutively transcribed in the opposite direction. This promoter system was designed as a tightly regulated, user modulated, inducible system and is suitable for use in a ‘kill switch’.
References:
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The AraC protein forms a dimer, each molecule binds to the araI1 and araO2 regulatory regions of the DNA and prevents the binding of RNA polymerase to the P-araBAD promoter region and hence represses the transcription of the downstream gene.
In the presence of Arabinose (and low amount of glucose sugar), the Arabinose binds to the AraC protein dimer, brings about a conformational change and binds to the regions araI1 and araI2 regions of the DNA releasing the P-araBAD promoter. Now, RNA polymerase binds to the P-araBAD promoter and proceeds with transcription. The incorporation of RNA polymerase at the P-araBAD promoter is enhanced by the CAP-cAMP complex bound to the CAP binding site. As the concentration of cAMP is inversely proportional to the concentration of glucose, for a constant amount of CAP protein, lesser the concentration of glucose, more will be the concentration of cAMP molecules, hence enhanced transcription of any downstream gene.
Figure 1: The designed arabinose inducible promoter system. The araC is constitutively transcribed in the opposite direction. This promoter system was designed as a tightly regulated, user modulated, inducible system and is suitable for use in a ‘kill switch’.
References:
- Guzman, Luz-Maria, et al. "Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter." Journal of bacteriology 177.14 (1995): 4121-4130
- https://parts.igem.org/Part:BBa_K2442101
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Sequence and Features
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1043
Illegal NheI site found at 1066 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1388
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1223
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1205
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Categories
Parameters
| None |