Composite
Part:BBa_K3519012
Designed by: Omkar Mohapatra Group: iGEM20_IISER-Tirupati_India (2020-10-07)
Revision as of 11:54, 24 October 2020 by Omkarmohapatra123 (Talk | contribs)
araC-araBAD promoter-DNASEI
This part contains the DNASEI gene downstream to the arabinose inducible promoter. The gene is the bovine pancreatic DNaseI isolated from Bos taurus. This is a tightly regulated 'kill switch'. The gene is expressed on the addition of arabinose to the media. Excess glucose in the media represses the expression of the endonuclease thus preventing cell death. All data presented below have been adapted from the literature.
Usage and Biology
The araC protein is constitutively produced by the medium strength Anderson promoter (J23118) and the strong RBS (B0034) and production of DNASE1 (bovine pancreatic DNASE1) is controlled by the P-araBAD.
Figure 1: The ‘native’ genetic circuit of the engineered ‘kill switch’. The araC is constitutively transcribed in the opposite direction.
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 DNASEI gene.
Figure 2: The ‘inactive’/repressed genetic circuit of the engineered ‘kill switch’. The system here is switched ‘off’ in absence of arabinose in the media. The araC gene is constitutively transcribed in the opposite direction. The AraC dimer binds to the regulatory regions of the promoter and represses the transcription of DNASEI.
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 DNASEI gene here.
Figure 3: The ‘active’ genetic circuit of the engineered ‘kill switch’. The system here is switched ‘on’ in addition of arabinose to the media. The araC is constitutively transcribed in the opposite direction. The promoter is activated on recruitment of the cAMP-CAP complex and the arabinose-AraC complex to the regulatory regions in the promoter, leading to increase in the transcription of the DNASEI gene.
References:
Figure 1: The ‘native’ genetic circuit of the engineered ‘kill switch’. The araC is constitutively transcribed in the opposite direction.
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 DNASEI gene.
Figure 2: The ‘inactive’/repressed genetic circuit of the engineered ‘kill switch’. The system here is switched ‘off’ in absence of arabinose in the media. The araC gene is constitutively transcribed in the opposite direction. The AraC dimer binds to the regulatory regions of the promoter and represses the transcription of DNASEI.
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 DNASEI gene here.
Figure 3: The ‘active’ genetic circuit of the engineered ‘kill switch’. The system here is switched ‘on’ in addition of arabinose to the media. The araC is constitutively transcribed in the opposite direction. The promoter is activated on recruitment of the cAMP-CAP complex and the arabinose-AraC complex to the regulatory regions in the promoter, leading to increase in the transcription of the DNASEI gene.
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
- Chen, C. Y., Lu, S. C., & Liao, T. H. (1998). Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme. Gene, 206(2), 181–184. https://doi.org/10.1016/s0378-1119(97)00582-9
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 |