Difference between revisions of "Part:BBa K3009033"

 
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<partinfo>BBa_K3009033 short</partinfo>
 
<partinfo>BBa_K3009033 short</partinfo>
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<b>Group:</b> Freiburg
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<b>Author:</b> Alisa
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<b>Summary:</b>  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.
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Documentation:
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===Usage===
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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]
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===Biology===
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In C. elegans, SPD5 , forms condensates that aid the organization of microtubule arrays in the centrosome. [2]
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Characterization
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By expressing a SPD5:sfGFP fusion in E.coli, we have proven droplet formation in the E.coli cytosol.
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[[File:BBa K3009033_1.png|200px|thumb|left|Fig. 1: E.coli cells expressing a SPD5:sfGFP fusion verify droplet formation]]
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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
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[[File:BBa K3009033 FRAP.mp4|200px|thumb|left|Fig.2: Fluorescence Recovery after Photobleaching of membraneless organelle]]
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===Referrences===
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[1]] Reinkemeier et. al 2019): Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes. In: Science (New York, N.Y.) 363 (6434).
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[2]Woodruff et. al (2017): The Centrosome Is a Selective Condensate that Nucleates Microtubules by Concentrating Tubulin. In: Cell 169 (6), 1066-1077
  
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Revision as of 03:12, 22 October 2019


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]