Part:BBa_K3447102

Arabinose production
XynA is a xylanase gene derived from Bacillus subtilis 168 and encodes 1, 4-endonuclide xylanase. XynD gene, derived from Bacillus subtilis 168, can specifically hydrolyze the Arabinose residues on the O-2 xylan or O-3 xylan, and assist the hydrolysis of the xylan.

Usage and Biology

In our project, we use them to hydrolyze xylan in culture medium to produce arabinose. For more information, please visit our Design page: https://2020.igem.org/Team:Jilin_China/Design

Characterization

To verify the construction of xynA-xynD, the digestion and agarose gel electrophoresis were performed by a standard protocol (Fig. 1A).
Moreover, Acidic Xylanase Assay (DNS demonstration method) was used to determine the enzyme activity of xylanase. As shown in Fig. 1B, the xylanase was produced as expected, and the enzyme activity is 0.105 U/mL (Definition of enzyme activity: 1 unit, the amount of enzyme required to hydrolysis xylan to produce 1 μmol of reducing sugar per milliliter of fermentation broth, under the condition of 50℃ and pH 4.8).

Fig. 1 Xylanase could be produced by xynA and xynD. (A) Digestion and electrophoresis of xynA-xynD. (B) The absorption curve of the xylose standard at 495 nm in DNS reaction. 5 g/mL xylan was added to the supernatant, which was centrifuged and collected in xylanase producing bacteria were incubated overnight in advance, and the enzyme activity was measured by DNS method at 50℃ for 30 mins. The concentration of xylose after hydrolyzation was determined by comparing to the standard curve.

Design

Design Notes

We added some synonymous mutations to avoid part rules.

Source

We found this sequence data in the previous iGEM part (BBa_K1175005) and in GenBank.

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