Part:BBa_K3009033

SPD5 Group: Freiburg Author: Alisa Summary: Spindle-deficient protein 5 derived from C.elegans is able to assemble into condensates that built a separate phase from the surronding cytosol, therefore emulating a reaction compartment. Fusion proteins will also be confined to this organelle. Documentation:

Usage

SPD 5 proteins assemble into phase-separated condensates in the cytosol, building a dynamic artificial organelle. Fusion proteins of SPD5 will be confined inside the organelle and separated from the all other components inside the cell. Due to the lack of a membrane, all other molecules can freely move in and out of the organelle.[1]

Biology

In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome. [2]

Characterization

By expressing a SPD5:sfGFP fusion in E.coli, we have proven droplet formation in the E.coli cytosol.

Fig. 1: E.coli cells expressing a SPD5:sfGFP fusion verify droplet formation

To confirm that the constructs were were seeing were in fact dynamic droplets and not dead aggregates, we bleached several regions of interest with a 475 nm laser beam. Due to the dynamic nature of the droplets, flurescent should be recovered by diffusion of unbleached proteins

File:BBa K3009033 FRAP.mp4

Referrences

[1]] Reinkemeier et. al 2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434). [2]Woodruff et. al (2017): The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. In: Cell 169 (6), 1066-1077

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 769
  Illegal AgeI site found at 1138
  Illegal AgeI site found at 1237
  
• 1000
  COMPATIBLE WITH RFC[1000]