Part:BBa_K3196002

SLAC

Small laccase gene

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 345
  

Characterization

Amplified from S. coelicolor.

Usage and Biology

Laccase is a multicopper oxidase, capable of catalyzing one-electron oxidation of a wide range of substrates to generate radicals while concomitantly reducing molecular oxygen to water^[1]. Laccases are widely distributed in nature. They have been isolated from fungi, plants, insects and bacteria^[2] of which fungal laccases are well characterized ^[3]. Bacterial laccases, such as the small laccase from S. coelicolor (SLAC), can be useful alternatives to fungal laccases. SLAC exhibits some desirable characteristics such as activity at high temperatures and alkaline pH ^[4], making it a potential candidate in biotechnological applications. Extracellular expression could be a desirable alternative for expression of foreign proteins since it simplifies purification steps ^[5]. P. pastoris is usually the preferred host for the production of industrial enzymes.  

DNA Gel Electrophoretic

Signal peptide FLO10, FLO10 αpro, PHO1, PHO1 αpro can increase the activity of laccase expressed in Pichia pastoris ^[6] . We chose 5 signal peptides and added on the upstream of SLAC, using AOX1 promoter to start gene expression and using his tag to purify SLAC enzyme. Assembled SLAC with different signal peptides on the one hand enabled us to measure the effects of enzymes, and on the other hand facilitated us to select specific combinations of signal peptides and enzymes to build system.

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.

Figure2:The image above shows the successful transformation of the target gene into the Pichia pastoris genome, including SLAC FLO10, SLAC PHO5 apro, and SLAC.

SDS-PAGE

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

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.