Part:BBa_K1031221

Po-B0031-sfGFP-Terminator (DmpR)

Structure

Po promoter which is activated by DmpR, is σ-54 dependent. It is composed of three regions. The two inverted UAS sites are responsible for binding of DmpR transcriptional factor. The two IHF binding sites allowing IHF to participate enhance transcription efficiency. -24 and -12 region interact with sigma-54 factor of RNA polymerase, enabling the formation of open complex. (Fig 1)

Fig 1 Po promoter structure. The UAS of this promoter marked in green is combined of two parts in contrast direction to which DmpR binds. The box with yellow background represents IHF binding sites. The box with pink background represents σ54 binding site with -24 region and -12 region marked in red. The G with right angle represents +1 site.

Sequence and Features

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 212
  

Construction and data

We constructed a library of RBS (Ribosome Binding Site) of different strength for tuning the expression of reporter sfGFP. K1031221 is a reporter circuit composed of three elements, the inducible promoter Pofor DmpR, RBS B0031[1], and reporter gene sfGFP. (Fig 2)

Fig 2 Construction of reporter circuit. The orange arrow represents Po promoter for DmpR. The green oval stands for RBS B0032. sfGFP coding sequence is shown with dark blue, while terminator B0015[2] is in dark red.

The performance of each reporter circuit adopting B0031, B0032, B0033 and B0034 in collocation with Po/DmpR circuit was tested.(Fig 3)

Fig 3Induction ratio of DmpR when exposed to a series of concentration of ***. The reporter circuit includes Po-RBS-sfGFP. Three lines represent sfGFP controlled by different RBS. Fluorescence intensity of sfGFP is detected and calculated to plot induction ratio