Difference between revisions of "Part:BBa K2271060"
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===Usage and Biology===
 
===Usage and Biology===
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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).
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[[File:Snc1duscgn.png|200px|thumb|right|'''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. [https://parts.igem.org/Part:BBa_K2271060:Design [1].]]]
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[[File:Snc1duscgn.png|200px|thumb|right|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)]]
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<p align="justify">
 
<p align="justify">
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This part is a truncated version of the v-SNARE ('''v'''esicle- synaptosome-associated-'''S'''oluble '''N'''-ethylmaleimide-sensitive-factor '''A'''ttachment '''RE'''ceptorprotein) 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 α-helical segments (forming the SNAREpin with the t-SNARE) and the transmembrane domain.[https://parts.igem.org/Part:BBa_K2271060:Design [1]]
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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).
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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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K2271103 Pex15] /[https://parts.igem.org/Part:BBa_K2271144 PEX26]) to secrete the compounds of this compartment.
  
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[[File: T--Cologne-Duesseldorf--SecretionSNAREpic.png|500px|thumb|center|'''Figure 2.''' Concept of secreting peroxisomal contents to the supernatant. For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.]]
  
 
===Experimental design===
 
===Experimental design===
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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).  
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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 (beta-glucuronidase)to perform a [http://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion GUS Assay]. 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===
 
===Results===
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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.  
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Different peroxisomal membrane anchors were tested using the GUS-assay. The highest activity of GUS could be measured in the supernatant of [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2271103 Pex15] as a membrane anchor.
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[[File:Igemduscgn2017GUSassay.png|500px|thumb|center|'''Figure 2.''' Relative fluorescence units per minute (RFU/min) measured for supernatants of different S. cerevisiae strains. The fluorescence was measured for 12 hours in intervals of 10 minutes with an excitation of 365 nm and an emission of 465 nm. For the strain BY4247 (wt) which was used as the background strain the fluorescence did not increase over measured period. The lysis controls (GUS-PTS1; ∆Pex11 GUS-PTS1) showing a lower activity than the samples of strain with peroxisomes decorated with Snc1. The highest activity could be measured in the strain using Pex15 with a linker as a membrane anchor (Pex15 L). The assay was performed in three technical replicates.]]
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2271060 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2271060 SequenceAndFeatures</partinfo>
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===References===
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[1] <b> Conserved α-Helical Segments on Yeast Homologs of the Synaptobrevin/VAMP Family of v-SNAREs Mediate Exocytic Function. </b> <br>
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Gerst, Jeffrey E. (1997) <br>
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<i>J. Biol. Chem. 272 (26), pp. 16591–16598.</i> DOI: 10.1074/jbc.272.26.16591.
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[2] <b>Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites. </b> <br>
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André Halbach, Christiane Landgraf, Stephan Lorenzen, Katja Rosenkranz, Rudolf Volkmer-Engert, Ralf Erdmann, and Hanspeter Rottensteiner (2006)
 +
In Journal of Cell Science 119, 2508-2517 Published by The Company of Biologists 2006 doi:10.1242/jcs.02979
  
  

Latest revision as of 00:28, 2 November 2017


Snc1


Usage and Biology

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. [1.]

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 α-helical segments (forming the SNAREpin with the t-SNARE) and the transmembrane domain.[1] 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 (Pex15 /PEX26) to secrete the compounds of this compartment.

Figure 2. Concept of secreting peroxisomal contents to the supernatant. For the secretion, the membrane anchor Pex15 or Pex26 is used. This anchor is used to decorate peroxisomes or our modified compartments with the v-SNARE Snc1. For the secretion Snc1 interacts with the t-SNAREs in the cell membrane. Induced from this interaction the vesicle and cell membrane fuse and the content of the compartment is secreted to the supernatant.

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 (beta-glucuronidase)to perform a [http://2017.igem.org/Team:Cologne-Duesseldorf/Design#Secretion GUS Assay]. 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 as a membrane anchor.

Figure 2. Relative fluorescence units per minute (RFU/min) measured for supernatants of different S. cerevisiae strains. The fluorescence was measured for 12 hours in intervals of 10 minutes with an excitation of 365 nm and an emission of 465 nm. For the strain BY4247 (wt) which was used as the background strain the fluorescence did not increase over measured period. The lysis controls (GUS-PTS1; ∆Pex11 GUS-PTS1) showing a lower activity than the samples of strain with peroxisomes decorated with Snc1. The highest activity could be measured in the strain using Pex15 with a linker as a membrane anchor (Pex15 L). The assay was performed in three technical replicates.



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]

References

[1] Conserved α-Helical Segments on Yeast Homologs of the Synaptobrevin/VAMP Family of v-SNAREs Mediate Exocytic Function.
Gerst, Jeffrey E. (1997)
J. Biol. Chem. 272 (26), pp. 16591–16598. DOI: 10.1074/jbc.272.26.16591.

[2] Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites.
André Halbach, Christiane Landgraf, Stephan Lorenzen, Katja Rosenkranz, Rudolf Volkmer-Engert, Ralf Erdmann, and Hanspeter Rottensteiner (2006) In Journal of Cell Science 119, 2508-2517 Published by The Company of Biologists 2006 doi:10.1242/jcs.02979