Part:BBa_K3196011

AOX1-Kozak-FLO10-SLAC-His tag-AOX1 Terminator

FLO10 is a Lectin-like protein with similarity to Flo1 p, thought to be expressed and involved in flocculation. SLAC can catalyze lignin.

Characterization

This is a four section for degrade and transfer lignin part.

Figure1. AOX1-FLO10-SLAC-His tag.

DNA Gel Electrophoretic

After we link FLO10 and SLAC successfully, we run the PCR with an intention to confirm the expression of SLAC. As the figure shows, we get the genetic stripe about 2009 bp which means the PCR is successful.

Figure1:This is the result of the SacⅠ single endonuclease digestion plasmid of the TOP10 strain, we will extract the gel and obtain the aimed DNA.

SDS-PAGE

We run the SDS-PAGE to check whether FLO10 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about 35 KDa which means the SDS-PAGE is successful.

Figure3:the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.

Enzyme Activity

We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity.

Figure4:these four pictures shows the enzyme activity changes with the time. The four kinds of Pichia pastoris have the different curve, and the most activity strain is FLO10-apro, PHO5apro shows a more delayed increase on enzyme activity.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 937
  Illegal BamHI site found at 948
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1387