Part:BBa_K3447102

Arabinose production

P_C is a constitutive promoter that expresses the downstream araC gene. P_BAD promoter is an inducible promoter, P_BAD contains sequences of P_C. It was verified by iGEM 2017 Amazonas_Brazil team that the induction effect of arabinose could be realized by using only araC and P_BAD.

Usage and Biology

Arabinose operon is composed of AraC protein, P_BAD and P_C. The araC gene translates from the P_C to the left.
We used the P_C promoter to express AraC constitutively, and constituted the sensing part of the arabinose sensing system.

AraC gene expression product belongs to araC protein family, which is a type of two monomers with dimer structure, and each monomer with DNA binding sites of the spiral screw - corner - functional areas. It’s amino terminal determines the inducibility and it’s carboxyl terminal determines the DNA binding ability. Through this helix-rotation-helix structure, araC contacts and binds to four large sulcus regions adjacent to the DNA.

Figure 1. Diagram of araC binding with α-L-Arabinose. (a)α-L-arabinose, α-D-arabinose and β-D-arabinose structures. (b) A wild-type AraC combined with L-Arabinose (LA) binds to the crystalline structure of the pocket. The amino acids identified here are residues that play an important role in ligand binding.

The dimerization domain is formed by folding into a pocket, combining arabinose, and dimerized by an antiparallel coiled coil. AraC stimulates RNA polymerase binding to DNA and the rate of formation of open complexes. The presence or absence of arabinose may alter the DNA contact of the upstream subunit of homodimer AraC, thereby affecting the binding of RNA polymerase. In the absence of L-arabinose, the DNA-binding domain (DBD) of the AraC dimer binds the I₁ and O₂ halves (separated by 210 bases) and inhibits transcription by forming a DNA ring upstream of the dimer. After P_BAD binds to L-arabinose, the dimer changes the conformation, making DBD bind to the adjacent I₁ and I₂ halves, thus leading to transcriptional activation through interaction with RNA polymerase in P_BAD. The regulatory characteristics of AraC are due to the sensitive conversion between the two conformation Pr and Pi, and the specific interaction with the inducer.

Figure 2. Schematic diagram of araBAD gene induced expression mechanism

Figure 3. AraC action mechanism diagram

In our project, we used this part together with P_C and P_BAD to form the arabinose sensing system, and achieved different functions by linking different target genes downstream of P_BAD.

Arabinose induces endotoxin expression: (链接a01+c07)
Arabinose induces green fluorescent protein expression: (链接a01+c09)

Design

Design Notes

We added some synonymous mutations to avoid part rules.

Source

We found this sequence data in the previous iGEM teams (Glasgow 2017) and in GenBank.

References

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 22
  Illegal NheI site found at 45
  Illegal NheI site found at 166
  Illegal NheI site found at 891
  Illegal NheI site found at 914
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 76
  Illegal BsaI.rc site found at 570
  Illegal BsaI.rc site found at 1657
  Illegal BsaI.rc site found at 1817
  Illegal SapI.rc site found at 624