Part:BBa_K3196012

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

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
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1586
  

The FLO10 was combined with the guiding peptide sequence of saccharomyces cerevisiae to form the signal peptide FLO10- αpro.FLO10 αpro is a combined signal peptide, which enhance the enzyme activity 3 times.

Characterization

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

Figure1. AOX1-FLO10-αpro-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 2208 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, PHO1apro shows a more delayed increase on enzyme activity.