Part:BBa_K4077008

Laccase from Melanocarpus albomyces gene insert

Laccase from Melanocarpus albomyces gene insert intended for pPIC9k.

Description

1.BBa_K3196036: AOX1 promoter region is a common inducible promoter from the Pichia pastoris genome. Alcohol oxidase is the major enzyme transcribed and converts methanol to formaldehyde. The part is made by iGEM19_HUST-China.
2.BBa_K4077005: The Alpha-factor secretion signal native to Saccharomyces cerevisiae is genetically engineered into Pichia pastoris expression vector. In order to secrete proteins for easier purification from the extracellular medium.
3.BBa_K4077000: The laccase was selected as our ideal enzyme through dry lab experiments, It was inserted into the expression vector.
4.BBa_K3196026: The terminator is used in conjunction with the AOX1 promoter. The part is made by iGEM19 HUST China
5.BBa_K3196026: Pichia wild-type gene coding for histidinol dehydrogenase (~2.4 kb) and used to complement Pichia his4 strains. Provides a selectable marker to isolate Pichia recombinant strains.

References

1.Lin-Cereghino, G.P., Stark, C.M., Kim, D., Chang, J., Shaheen, N., Poerwanto, H., Agari, K., Moua, P., Low, L.K., Tran, N., Huang, A.D., Nattestad, M., Oshiro, K.T., Chang, J.W., Chavan, A., Tsai, J.W. and Lin-Cereghino, J. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris. Gene, [online] 519(2), pp.311–317. Available at: http://www.sciencedirect.com/science/article/pii/S0378111913001765.
2.. Mayer, A. M. & Staples, R. C. (2002). Laccase: new functions for an old enzyme. Phytochemistry, 60, 551–565.
3.Hakulinen, N., Kiiskinen, L.-L., Kruus, K., Saloheimo, M., Paananen, A., Koivula, A. & Rouvinen, J. (2002). Crystal structure of a laccase from Melanocarpus albomyces with intact trinuclear copper site. Nat. Struct. Biol. 9, 601–605.
4.Kiiskinen, L.-L., Viikari, L. & Kruus, K. (2002). Purification and characterization of a novel laccase from the ascomycete Melanocarpus albomyces. Appl. Microbiol. Biotechnol. 59, 198–204
5.(User Manual PPIC9K a Pichia Vector for Multicopy Integration and Secreted Expression Catalog No. V175-20)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 3180
  Illegal PstI site found at 959
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 3888
  Illegal PstI site found at 959
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2233
  Illegal BglII site found at 2536
  Illegal XhoI site found at 1180
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 3180
  Illegal PstI site found at 959
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 3180
  Illegal PstI site found at 959
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 5228
  Illegal BsaI.rc site found at 2262