Part:BBa_K4229077

SPD5 with snoopTag&spyTag +RBS

This Biobrick is the improved Biobrick BBa_K4229076 with his RBS BBa_K4229039. Self-assembling proteins such as the spindle-deficient protein 5 (SPD-5) derived from Caenorhabditis elegans can form spontaneously, assembling dynamic organelles in vitro and in vivo [1]. Not only does SPD-5 reliably form droplet-like structures even in the highly crowded environment of the cytoplasm, but it also has been shown to naturally recruit enzymes and related molecules into the dynamic formation[2][3]. SPD-5 contains nine predicted coiled-coil domains, which make up ~40% of the protein. Studies have shown that the coiled-coil domains are involved in the formation of stoichiometric protein complexes and drive the formation of amorphous condensates followed by a rearrangement stage. SPD-5 condensates form as viscous liquids that quickly harden [2]. Even though SPD-5 is dynamic and does not permit exclusive entry and exit of specific molecules, it has been successfully used to enhance the efficiency of reactions, for example, by improving non-canonical amino acid incorporation with an orthogonal translation system ([4] and iGEM Freiburg 2019). In order to target cargos specifically to liquid droplets the SpyCatcher/SpyTag and SnoopCatcher/SnoopTag systems (explained above) was applied to enhance the property SPD-5 based compartments.

[1] D. R. Hamill, A. F. Severson, J. C. Carter, and B. Bowerman, “Centrosome maturation and mitotic spindle assembly in C. elegans require SPD-5, a protein with multiple coiled-coil domains,” Dev. Cell, vol. 3, no. 5, pp. 673–684, 2002, doi: 10.1016/S1534-5807(02)00327-1.

[2] J. B. Woodruff, B. Ferreira Gomes, P. O. Widlund, J. Mahamid, A. Honigmann, and A. A. Hyman, “The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin,” Cell, vol. 169, no. 6, pp. 1066-1077.e10, 2017, doi: 10.1016/j.cell.2017.05.028.

[3] A. K. Tiwary and Y. Zheng, “Protein phase separation in mitosis,” Curr. Opin. Cell Biol., vol. 60, no. 1, pp. 92–98, Oct. 2019, doi: 10.1016/j.ceb.2019.04.011.

[4] C. D. Reinkemeier, G. E. Girona, and E. A. Lemke, “Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes,” Science (80-. )., vol. 363, no. 6434, 2019, doi: 10.1126/science.aaw2644.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2405
  Illegal EcoRI site found at 3579
  Illegal SpeI site found at 1383
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2405
  Illegal EcoRI site found at 3579
  Illegal SpeI site found at 1383
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2405
  Illegal EcoRI site found at 3579
  Illegal BglII site found at 2306
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2405
  Illegal EcoRI site found at 3579
  Illegal SpeI site found at 1383
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2405
  Illegal EcoRI site found at 3579
  Illegal SpeI site found at 1383
  
• 1000
  COMPATIBLE WITH RFC[1000]