Part:BBa_K3447104

Arabinose induces endotoxin expression

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.

Usage and Biology

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.

Characterization

The digestion and agarose gel electrophoresis are shown in Figure 1.

Fig. 1 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. 2A and 2B 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. 2C).

Fig. 2 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.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1239
  Illegal BsaI.rc site found at 1705
  Illegal SapI site found at 961