Part:BBa_K3447001

Arabinose sensing system

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 BBa_I0500 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.
As shown in Figure 1 and 2, when arabinose is present in cells, araC, the product encoded by gene araC, binds to arabinose and binds to the upstream promoter region of P_BAD to activate RNA polymerase and activate the gene after P_BAD. On the contrary, when arabinose is not present, the product araC will cause DNA curling to inhibit the binding of RNA polymerase, and inhibit the expression of gene downstream of P_BAD.

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.

Figure 2. Schematic diagram of arabinose operon regulation

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 3. Schematic diagram of araBAD gene induced expression mechanism

Figure 4. 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: BBa_K3447104
Arabinose induces green fluorescent protein expression: BBa_K3447103

Characterization

Validation of Arabinose response system

• See details in BBa_K3447103.
  

This part contains an arabinose operon, which constitutes the arabinose sensing system. By connecting the sfGFP gene downstream, we were able to verify the expression intensity of this arabinose operon.

To prove the arabinose sensing system can work successfully, a sfGFP was inserted after P_BAD under the induction of arabinose at a series of concentrations. As shown in Fig. 5B, the expression efficiency was highest when induced with 0.2 g/mL arabinose.

Fig. 5 Arabinose can induce the downstream expression. (A) Digestion and electrophoresis of araC-P_BAD-sfGFP. (B) AraC-PBAD induction at different concentrations of arabinose. E. coli DH5α was transformed with the designed plasmid, cultured for 8 hrs, and diluted OD600 to 0.04. 0.02, 0.2 or 2 g/mL arabinose was added respectively. The fluorescence intensity of sfGFP was detected at the indicated time. The experiment was performed three times in triplicate. *, P < 0.05 from respective control using Student’s t test.

Arabinose induces endotoxin expression

• See details in BBa_K3447104.
  

This part contains an arabinose operon, which constitutes the arabinose sensing system. By connecting the relE gene downstream, the normal growth and reproduction of bacteria will be inhibited when arabinose is present.

After the expression of arabinose operon was verified, sfGFP gene was replaced by relE gene, which causes the normal growth and reproduction of bacteria will be inhibited when there is arabinose in the medium.

The digestion and agarose gel electrophoresis are shown in Fig. 6.

Fig. 6 Digestion and electrophoresis of araC-P_BAD-relE

Since relE encodes a small endotoxin peptide which represses the growth of bacteria, its expression could be characterized by measuring growth curve. Fig. 7A and 7B suggest that, compared with negative control, the E. coli cells cultured with standard arabinose can be inhibited for growth and reproduction. Finally, in order to verify whether the xylanase produced by E. coli could manage to hydrolyze xylan to arabinose and, by binding to the araC, turn on the expression of relE in the downstream of P_BAD, thus proving its function by measuring the growth situation (Fig. 7C).

Fig. 7 RelE was induced to inhibit bacterial growth. The growth curve of E. coli DH5α with original OD₆₀₀ value of (A) 0.1 (B) 0.8 (C) 0.3. The standards of gradient concentration (A and B) and xylanase-containing bacteria supernatant which had been pre-cultured overnight (C) were added into the medium. The supernatant of fermentation broth containing xylanase was isolated and added into the E. coli culture system. Then, 5 g/mL xylan was added respectively for being reduced by xylanase and measure OD₆₀₀ values at the indicated time. The experiment was performed three times in triplicate. ***, P < 0.001 from respective control using Student’s t test.

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 BBa_I0500) and in GenBank.

References

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1216
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1155
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 990
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1
  Illegal BsaI.rc site found at 1245
  Illegal SapI site found at 972