Coding

Part:BBa_K2271060

Designed by: Philipp Rink and Jan Maika   Group: iGEM17_Cologne-Duesseldorf   (2017-10-27)
Revision as of 22:01, 29 October 2017 by Jan-maika (Talk | contribs)


Snc1


Usage and Biology

This part is a truncated version of the v-SNARE (vesicle- synaptosome-associated-Soluble N-ethylmaleimide-sensitive-factor Attachment REceptorprotein) Snc1. Snc1 is in the wildtype form involved in the fusion of Golgi-derived secretory vesicles with the plasma membrane. Domains of the protein are a variable domain which is not important for the binding to the t-SNARE, H1 and H2 are the a-helical segments (forming the SNAREpin with the t-SNARE) and the transmembrane domain (Gerst Paper).

Figure 1. A diagram of the general domain structure of Snc1. V is a variable domain which is not important for the binding to the t-SNARE. TM is the transmembrane domain. H1 and H2 are the α-helical segments, forming the SNAREpin with the t-SNARE Gerst et al. (1997)
For our approaches we used a truncated version without the transmembrane domain. This Snc1 truncation was fused to the N-Terminus of different peroxisomal membrane anchor (Link Pex15/Pex26) to secrete the compounds of this compartment (Bild).



Experimental design

For testing this part we used a fusion with the N-Terminus of a peroxisomal membrane anchor. We co-expressed this construct with GUS-PTS1 to perform a GUS Assay.(oder: We performed a GUS-Assay by targetting GUS(beta-Glucuronidase) to the peroxisome using a GUS-PTS1.) The secreted GUS in the supernatant was measured with the turnover of 4-methylumbelliferyl-beta-D-glucuronide to 4-methyl umbelliferone (4-MU). The fluorescent 4-MU was measured with a plate reader (excitation: 365 nm, emission: 465 nm).

Results

Different peroxisomal membrane anchors were tested using the GUS-Assay. The highest activity of GUS could be measured in the supernatant of Pex15 (Link to part) as a Membrane Anchor.


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
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None