Part:BBa_K3165057
T7 - nMag + T7 - pMag + mOrange (for Bacillus subtilis)
This composite system consists of the entire optogenetic T7 RNA Polymerase system with mOrange under the PcpcG2 promoter.
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 site found at 828
Illegal BsaI site found at 1723
Illegal BsaI.rc site found at 2257
Illegal BsaI.rc site found at 2296
Usage and Biology
The T7 DNA-dependent RNA Polymerase is commonly used for protein expression due to its high processivity and high selectivity for T7 promoter. Bacterial RNA Polymerase cannot transcribe the genes under the T7 promoter, so under normal circumstances, the genes under the T7 promoter will not be transcribed and hence the gene product won't be synthesized (some lysogenic bacterial strains can produce the proteins under T7 expression).
Thus we can use the T7 expression system in our model organism for protein expression. Splitting up of the T7 RNA Polymerase into two separate domains, each linked to a photo-sensitive dimerizing unit is an effective means to regulate protein expression in a cell. Upon shining light of appropriate frequency (blue light for the T7 system), the photo-sensitive domains dimerize leading to the functional reactivity of the T7 RNA Polymerase. Upon stimulation, the T7 RNA Polymerase becomes functional and transcribes the genes downstream to the T7 promoter, providing a dynamic means to control protein expression.
This part uses mOrange fluorescent protein, for characterising the functional activity of the composite system. This part encodes for the entire opto T7 RNA Polymerase system with mOrange as the reporter gene for the light-mediated regulation of protein expression.
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