Part:BBa_K3165009

C-Terminal T7 RNAP Domain + pMag (Optimised for B. subtilis)

This part consists of the C-Terminal T7 RNA Polymerase domain attached to a magnet domain (pMag) via a linker sequence. The T7 RNA Polymerase holoenzyme has been cleaved at specific sites to create two domains (the N-terminal T7 RNAP domain and the C-terminal T7 RNAP domain) both of which are individually linked to light-sensitive domains nMag and pMag via short linker sequences. On appropriate stimulation, the light-sensitive domains dimerize making the T7 RNA Polymerase functional again leading to the transcription of the genes under the T7 promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.

Usage

We can use this part in association with the other T7 domain (N-terminal domain) linked to the photo-sensitive nMag unit (BBa_K3165008) to create a blue light-sensitive system in the bacteria. The Opto T7 system to be incorporated into Bacillus subtilis utilises this part for its functioning.

References :

(1) "Dynamic blue light-inducible T7 RNA polymerases (Opto-T7RNAPs) for precise spatiotemporal gene expression control". Department of Biosystems Science and Engineering (D-BSSE), ETH–Zürich. doi:10.1021/acssynbio.7b00169