Difference between revisions of "Part:BBa K4444015"
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Our project builds on the protein circuit RELEASE (Vlahos et al.) by introducing light-inducible proteins or photoswitches. Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.). | Our project builds on the protein circuit RELEASE (Vlahos et al.) by introducing light-inducible proteins or photoswitches. Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.). | ||
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Luciferase is an enzyme that fluoresces when it binds with luciferin. By running a luciferase reporter assay with a luminometer on the media, one can quantitatively observe how much luciferase was secreted into the media. | Luciferase is an enzyme that fluoresces when it binds with luciferin. By running a luciferase reporter assay with a luminometer on the media, one can quantitatively observe how much luciferase was secreted into the media. | ||
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| + | Mechanism: Upon light-activation PhoCl breaks, removing the ER retention motif and allowing the protein to continue along the secretion pathway. The furin cut site will later be cleaved at the Golgi body, allowing the POI to be freely floating in the vesicle. At the plasma membrane the POI will be secreted. | ||
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Revision as of 08:15, 12 October 2022
PhoCl Secrete Luciferase
PhoCl Secrete Luciferase is modified from the RELEASE system. It contains a luciferase as a protein of interest, a furin cut site, PhoCl2c, tri-transmembrane domain, and an ER retention motif. PhoCl Secrete will completely release a protein of interest in response to ultra violet light stimulation.
Usage and Biology
Our project builds on the protein circuit RELEASE (Vlahos et al.) by introducing light-inducible proteins or photoswitches. Photocleavable protein (PhoCl) is a recently developed optogenetic technology that self-cleaves when stimulated by 405 nm ultraviolet light. PhoCl was engineered from a green-to-red photoconvertible fluorescent protein. When stimulated with ultraviolet light, the protein undergoes a β-elimination reaction, producing a small peptide fragment and a large barrel fragment that will spontaneously dissociate (Zhang et al.).
Luciferase is an enzyme that fluoresces when it binds with luciferin. By running a luciferase reporter assay with a luminometer on the media, one can quantitatively observe how much luciferase was secreted into the media.
Mechanism: Upon light-activation PhoCl breaks, removing the ER retention motif and allowing the protein to continue along the secretion pathway. The furin cut site will later be cleaved at the Golgi body, allowing the POI to be freely floating in the vesicle. At the plasma membrane the POI will be secreted.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 52
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 991
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1162
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 604
Illegal BsaI.rc site found at 1684