Difference between revisions of "Part:BBa K2332001"
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<partinfo>BBa_K2332001 short</partinfo> | <partinfo>BBa_K2332001 short</partinfo> | ||
| − | This composite part consists of a blue-light inducible promoter (Pblind) upstream the gene encoding green fluorescence (GFP) codon optimized for E. coli. | + | This composite part consists of a blue-light inducible promoter (Pblind) upstream the gene encoding green fluorescence (GFP) codon optimized for E. coli. For blue-light transcriptional induction, cells must also express EL222 ([https://parts.igem.org/Part:BBa_K2332004 BBa_K2332004]). |
Pblind consists of a fusion of the EL222 DNA binding region and the LuxI promoter. The lux box, a 20bp inverted repeat (LuxR and 3-oxo-C6-HSL complex binding region) from the luxI promoter, was replaced with the 18bp DNA binding region of EL222, a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its binding region, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. In darkness, EL222 will reverse to its repressed state spontaneously. This optogenetic tool allows a dynamic, rapid and switchable transcriptional control. | Pblind consists of a fusion of the EL222 DNA binding region and the LuxI promoter. The lux box, a 20bp inverted repeat (LuxR and 3-oxo-C6-HSL complex binding region) from the luxI promoter, was replaced with the 18bp DNA binding region of EL222, a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its binding region, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. In darkness, EL222 will reverse to its repressed state spontaneously. This optogenetic tool allows a dynamic, rapid and switchable transcriptional control. | ||
This biobrick allowed us to test the effectiveness of the Pblind promoter by measuring the basal transcription of GFP in the absence of the blue light transcriptional activator EL222. | This biobrick allowed us to test the effectiveness of the Pblind promoter by measuring the basal transcription of GFP in the absence of the blue light transcriptional activator EL222. | ||
Revision as of 23:52, 22 October 2017
Blue light inducible expression system with GFP reporter (Pblind GFP)
This composite part consists of a blue-light inducible promoter (Pblind) upstream the gene encoding green fluorescence (GFP) codon optimized for E. coli. For blue-light transcriptional induction, cells must also express EL222 (BBa_K2332004). Pblind consists of a fusion of the EL222 DNA binding region and the LuxI promoter. The lux box, a 20bp inverted repeat (LuxR and 3-oxo-C6-HSL complex binding region) from the luxI promoter, was replaced with the 18bp DNA binding region of EL222, a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is inactive as its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its binding region, overlapping the -35 region of the luxI promoter. This ultimately results in the recruitment of RNAP and transcriptional activation. In darkness, EL222 will reverse to its repressed state spontaneously. This optogenetic tool allows a dynamic, rapid and switchable transcriptional control. This biobrick allowed us to test the effectiveness of the Pblind promoter by measuring the basal transcription of GFP in the absence of the blue light transcriptional activator EL222.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 709