Part:BBa_K3071013

PspA promoter

Description

This is an regulatory element that originated from Escherichia coli K12 strain. This basic part is used to construct our composite part (BBa_K3071014). Our rt-qPCR data show that this promoter is able to initiate the transcription in form of synthetic construct.

Biology

Figure 1 Basic properties of pspA promoter

pspA promoter is a sigma-54 (σ-54) regulated activator dependent promoter. In the orginal pspA promoter upstream region, it contains σ-54 consensus sequence 5' of the start contains a GG doublet as -24 and a consensus GG doublet at -12, the high-affinity IHF site (-25 to -60), as well as the UAS sites (UAS I: -89 to -107; UAS II:-111 to -129).

Usage

Figure 2 The transcription initiation mechanism of the sigma-54 promoters [http://www.cchem.berkeley.edu/wemmer/research/sigma54.html]

sigm54-RNA holoenzyme (σ-54 RNAP) forms an inactive transcriptional initiation complex on this promoter, which can be activated in E. coli by the bacterial enhancer-binding protein PspF (BBa_K3071006). PspF functions by binding to the upstream activation sequences (UAS) near the promoter and contacting the promoter-bound σ-54 RNAP via DNA looping stabilized by the binding of integration host factor (IHF). Previous research has demonstrated the property of pspF-dependent and enhancer-specific transcription activation of pspA promoter.

Characterization

Figure 3 illustration of our synthetic biological system upon DSF activation

The UAS sites of this promoter are replaced by CBS I(BBa_K3071011) and CBSII (BBa_K3071012) in our own synthetic system to construct CBS I & II-regulated pspA promoter (BBa_K3071014).

Figure 4 rt-qPCR data DSF treatment on the eforRED mRNA expression level

The rt-qPCR data upon activation by DSF indicate a significant change in gene expression, suggest the related composite part is functional.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 12
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]