Difference between revisions of "Part:BBa K2271103"

Revision as of 18:22, 30 October 2017

pex15

Biology and Usage

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)

This part contains a truncated version of Pex15 (315-383) from S. Cerevisiea . Halbach et al. (2006)
Pex15 is a tail anchord peroxisomal membrane protein with its c-terminus directed to the peroxisomal lumen and its n-terminus directed to the cytosol. It is required for peroxisome biogenesis. In cells lacking Pex15 peroxisomal matrix proteins mislocalize to the cytosol while overexpression results in impaired peroxisome assembly. Elgersma Y, et al. (1997)

Experimental design

The functionality of this part was validated by microscopy. For the microscopy mVenus was fused to the C-Terminus of Pex15. We could observe a peroxisomal typical localisation in the cell.
Additionaly we tested this part as a membrane anchor for the v-SNARE Snc1. We co-expressed the Snc1-Pex15 construct with GUS-PTS1 to perform a 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).

Figure 2. The micrscopy showing an localisation of the fluorescence protein mVenus with the typical peroxisomal shape. This indicates, an peroxisomal membrane licalisation of mVenus-Pex15

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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 Additionaly we tested this part as a membrane anchor for the v-SNARE [https://parts.igem.org/Part:BBa_K2271060 Snc1]. We co-expressed the Snc1-Pex15 construct with GUS-PTS1 to perform a 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).
-[[File:Igemduscgn2017GUSassay.png|500px|thumb|center|'''Figure 3.''' 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.]]
+[[File:Igemduscgn2017GUSassay.png|500px|thumb|center|'''Figure 2.''' The micrscopy showing an localisation of the fluorescence protein mVenus with the typical peroxisomal shape. This indicates, an peroxisomal membrane licalisation of mVenus-Pex15]]
 <!-- Add more about the biology of this part here
 ===Usage and Biology===