Difference between revisions of "Part:BBa K2271066"
 
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<partinfo>BBa_K2271066 short</partinfo>
 
<partinfo>BBa_K2271066 short</partinfo>
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===Usage and Biology===
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This is a composite part containing the fluorescent protein mRuby targeted to the peroxisome via an enhanced PTS1 described by DeLoache <i>et al.</i> (2016)[https://parts.igem.org/Part:BBa_K2271066:Design [1].]. PTS1 is a peroxisomal targeting signal recognized by a receptor called Pex5. Naturally, PTS1 consists of Ser-Lys-Leu (SKL) at the carboxy-terminus. Proteins harboring this C-terminal signal are imported into the peroxisome. The ePTS1 leads to an enhanced import of the taged protein.
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This part is designed as a peroxisomal marker for <i>S. cerevisiea</i>.[https://parts.igem.org/Part:BBa_K2271066:Design [1].] For fluorometric and microscopic applications the optimal excitation of 559 nm and emission of 600 nm is discriped.
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=== Experimental Design and Results===
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<p> For the validation the <i>S. cerevisiae</i> Strain BY4742 was tranformed with this part. The cells were fixated and microscoped with an Elyra PS microscope. A typical peroxisomal localisation could be validated (Figure 1).
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[[File:Igemducgn2017mRuby-PTS1.png|500px|thumb|center|'''Figure 1''' mRuby fused to the ePts1 described by DeLoache et al. (2016) [https://parts.igem.org/Part:BBa_K2271066:Design [1].] The mRuby is localised in the peroxisomes of the cells]] </p>
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The part contains a mRuby fluorezenz protein with an enhanced pts1 sequence, described by Dueber et. Al, with a TDH3 Promotor and a HHF1 Terminator. mRuby is a mutant oft he fluorezent protein gfp with excitation and emission wavelengths at 559 nm/600nm  . With the C-terminal enhanced pts1 (peroxisomal targeting sequnce) the mRuby is imported into the peroxisome. The part can be used as a peroxisomal matrix marker in <i>S. cerevisiea.</i>
 
  
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===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2271066 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2271066 SequenceAndFeatures</partinfo>
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===References===
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[1] <b>Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways </b> <br>
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William C. DeLoache, Zachary N. Russ & John E. Dueber <br>
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Nat. Commun. 7:11152 doi: 10.1038/ncomms11152 (2016).
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[2] <b>A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly </b> (2015)  <br>
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Michael E. Lee, William C. DeLoache, Bernardo Cervantes, and John E. Dueber <br>
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ACS Synth. Biol., 2015, 4 (9), pp 975–986 DOI: 10.1021/sb500366v
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Latest revision as of 22:22, 1 November 2017


mRuby-ePTS1

Usage and Biology

This is a composite part containing the fluorescent protein mRuby targeted to the peroxisome via an enhanced PTS1 described by DeLoache et al. (2016)[1.]. PTS1 is a peroxisomal targeting signal recognized by a receptor called Pex5. Naturally, PTS1 consists of Ser-Lys-Leu (SKL) at the carboxy-terminus. Proteins harboring this C-terminal signal are imported into the peroxisome. The ePTS1 leads to an enhanced import of the taged protein. This part is designed as a peroxisomal marker for S. cerevisiea.[1.] For fluorometric and microscopic applications the optimal excitation of 559 nm and emission of 600 nm is discriped.

Experimental Design and Results

For the validation the S. cerevisiae Strain BY4742 was tranformed with this part. The cells were fixated and microscoped with an Elyra PS microscope. A typical peroxisomal localisation could be validated (Figure 1).

Figure 1 mRuby fused to the ePts1 described by DeLoache et al. (2016) [1.] The mRuby is localised in the peroxisomes of the cells



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 860
    Illegal BamHI site found at 1577
    Illegal XhoI site found at 1613
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

[1] Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways
William C. DeLoache, Zachary N. Russ & John E. Dueber
Nat. Commun. 7:11152 doi: 10.1038/ncomms11152 (2016).


[2] A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly (2015)
Michael E. Lee, William C. DeLoache, Bernardo Cervantes, and John E. Dueber
ACS Synth. Biol., 2015, 4 (9), pp 975–986 DOI: 10.1021/sb500366v