Part:BBa_K1031021

Hybrid Promoter for Band-pass Filter

hybrid promoter for the band-pass filter

Construction of our hybrid promoter

We modified a bacteriophage ϕR73’s P₂ promoter into a hybrid promoter that can be activated by the ϕR73δ activator and repressed by the repressor cI simultaneously and put reporter sfGFP under its regulation. We constructed the hybrid promoter by replacing the sequence between position -1 and -25 of P₂ promoter with the cI binding site OR1 from Phage λ P_R promoter. When ϕR73δ activator binds to its target sequence upstream of -35 element of the hybrid promoter, the transcription will start. The binding of cI dimers downstream of -35 element will block the binding of σ⁷⁰ factors and thus repress the transcription even when ϕR73δ is bound. (Fig. 1).

Figure.1 Construction of Our Hybrid Promoter.
Sequence information of phage φR73 P₂ promoter (a), phage λ P_R promoter (b) and our hybrid promoter (c) are shown. a, In the P₂ promoter, φR73δ binds to a region between position -42 and -71 and activates transcription. b, In P_R promoter, cI dimer binds to OR1 site (marked as blue) between position -9 and -25, blocking binding of σ⁷⁰ factors and inhibiting transcription. cI binding region indicates the sequence we used to replace the corresponding region in P₂ promoter.c, The hybrid promoter is constructed by replacing sequence between position -1 and -25 of φR73 P₂ promoter with sequence at the same position in phage λ P_R promoter that contains an OR1 site. The hybrid promoter is co-regulated by φR73δ and cI, with φR73δ activating and cI repressing. The repression of cI dominates over the activation of φR73δ, since the steric hindrance created by cI dimer prevents formation of transcription initiation complex even when RNA polymerases are recruited through the help of φR73δ.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 127