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: The BBa_K2457000 includes approximately 1298 nucleotides and is used to regulate transgene expression in Escherichia coli bacteria. This construction is composed by the araBAD (PBAD) promoter, a medium force promoter, 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, transcribes the AraC gene in the opposite direction. AraC regulates the promoter’s function (Figure 1). In L-Arabinose absence, AraC dimerizes and binds itself in O2 and I1 operators, making a DNA “loop” around 210bp and avoiding the CAP-cAMP binding. Then it connects in a binding site upstream to 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 the L-Arabinose induces very low levels of PBAD transcription.

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

Although the L-Arabinose quantity may vary accordingly your expression experiment, the suggested L-Arabinose quantity is between 0,00002% and 0,2%.

Characterization

BBa_K2457000 was used by Amazonas_Brazil team in CROUT experiments and CRIN experiments.

CROUT (CRISPeasy-out method): Designed for knock-out strategy for bacterial genome editing based on CRISPR/Cas9

RESULTADOS DE CROUT AQUI JU

CRIN (CRISPeasy-in method): Designed for knock-in strategy for bacterial genome editing based on CRISPR/Cas9

RESULTADOS DE CRIN AQUI JU

Sequencing

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