Part:BBa_K3819088

XynB - GS linker - CtDockerin - His

The xylanase plasmid expresses the xylanase with Type1 dockerin that can be secreted to the outside of the cell, and the xylanase is connected to the dockerin through a flexible linker (GGGGS)3. The Type1 dockerin derived from from C. thermocellum can be combined with the Type1 cohesion on the scaffoldin to anchor the xylanase protein on the scaffoldin. In addition, there should be a tag on the xylanase plasmid. The short peptide expressed by the tag can be combined with the primary antibody and the secondary antibody with fluorescent dye to verify the binding of the cellulase to the backbone in the follow-up.

Finally, we successfully expressed this recombinant plasmid and detected the activity of xylanase XynB, which means XynB can work normally. And the results are as follows.

Figure 1. Dot Blot analysis of XynB. The sample was gained after fermentation in YPD for 96h. XynB(His-Tag) was tested by adding 2ul fermentation supernatant on NC membrane and exposing 60s.

Figure 2. The SDS-PAGE results of recombined GS115(pXynB) fermentation broths. Line 1 and 2 are GS115(pXynB) supernate and GS115 supernate respectively after 75-fold concentration. And at 43.4KDa, which is XynB with Ctdockerin's size, the expected band is not clear enough by naked eyes.

Figure 3. The Western-Blot results of recombined GS115(pXynB) fermentation broth. Control is GS115's concentrated fermentation supernatant. XynB is 43.4KDa. Line 1, 2, 3 refer to sediment, supernatant and concentrated supernatant.

Figure 4. The DNS results of XynB reflects enzymes' activity. The solution was gained after 96h of fermentation and concentrated by ammonium sulfate deposition method. 2ml enzyme solution reacted with 2ml 1% Xylan solution for 30min, then added 5ml DNS to shut down the process. During boiling in water bath, DNS combined with reducing sugar and produced dark brown, while control just shown unobvious color change.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]