Part:BBa_K3165054

(C-Terminal T7 RNAP Domain + pMag + mOrange) Generator (for Bacillus subtilis)

This part encodes for the C-terminal T7 RNA Polymerase domain attached to the pMag photo-sensitive unit via linker sequence with ECFP as a reporter gene (under pCpcG2 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]

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 a GFP derived fluorescent protein, ECFP to quantify the activity of the light-induced opto T7 system.
This part has the C-terminal T7 RNAP and pMag protein under a strong RBS forming a functional translational unit. We can use this part in association with the other T7 domain (N-terminal domain) linked to the photo-sensitive nMag unit 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.