Part:BBa_K2457000

araC + pBAD arabinose inducible promoter

Arabinose inducible promoter pBAD forward + araC regulatory reverse gene under control of pC promoter and tryptophan terminator sequence.

Figure 1: BBa_K2457000 Circuit.

Usage and Biology

Inductor: L-Arabinose

Repressor: AraC acts like repressor to both promoters (PBAD e PC) in glucose presence or L-Arabinose absence.

The BBa_K2457000 includes approximately 1298 nucleotides and is used to regule transgene expression in Escherichia coli bacterias. This construction is composed by the araBAD (PBAD) promoter, which produces medium force, the L-Arabinose operon, in 5’ → 3’ direction with a upstream native RBS, two regulatory sites: I1 and I2, and two operators sites AraO, O1 and O2. It is composed also by the promoter PC that transcribes the regulatory protein AraC gene, followed to tryptophan transcription terminator, in 3’ → 5’ (reverse) (KUHLMAN & COX, 2010).

The PC Promoter which is adjacent to PBAD promoter, it transcribes the AraC gene in the opposite way. AraC regulates the promoters activities (Figure 1). In L-Arabinose absence Arac dimerizes while binds itself in O2 and I1 operators, making a DNA “loop” around 210bp and avoids the CAP-cAMP binding, connect in a binding site upstream the I1 and I2 operators and RNA polymerase, that normally activate the transcription from PBAD and PC promoters (ENGLESBERG et al, 1965, YANOFSKY et al, 1981). In the presence of L-Arabinose, the pBAD expression is activated, meanwhile in the L-Arabinose absence produces very low levels of PBAD transcription.

The begin of transcription by PBAD only works in L-Arabinose presence and low glucose. L-Arabinose binds itself to AraC protein and the AraC N-terminal arm is released from your DNA binding domain through a “dimerization” mechanism. AraC changes its conformation and connects on I1 and I2 operators, this allows the CAP access to their binding site and it helps to recruit the RNA polymerase to the PBAD e PC promoters and then activate the gene transcription (Figure 2) (GUZMAN et al, 1995).

Characterization

Figure 2:Sequencing electropherogram from BBa_K2457000.

Figure 3:Alignment of the designed sequence and our final construction from BBa_K2457000.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 1207
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1042
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1024