Difference between revisions of "Part:BBa K2403003"
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===Usage and Biology===
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==Usage and Biology==
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In eukaryotic cells, it is known that RNA is transcribed in the nucleus and then exported to the cytoplasm, a process mediated by nuclear export factors specific to each type of RNA. In the case of single stranded mRNA, the TAP / p15 complex binds and exports it to the cytoplasm. However, artificial RNAs designed for synthetic biology applications are often highly structured RNA which are not recognized by TAP / p15, and therefore a different pathway is required to export such target RNAs to the cytoplasm.
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Aiming for feeding RNAi of <i>B. xylophilus</i>, our team had to confirm that <i>B. xylophilus</i> surely eats <i>S. cerevisiae</i>. To confirm this, we expressed high levels of EGFP in <i>S. cerevisiae</i> by combining '''[https://parts.igem.org/Part:BBa_K530008 BBa_K530008(TDH3 promoter)]''' and '''[https://parts.igem.org/Part:BBa_K1875003  BBa_K1875003 (EGFP)]''' and creating '''[https://parts.igem.org/Part:BBa_K2403003 BBa_K2403003]'''. Since this part produces a very strong green fluorescence, it is useful for tracking yeast.
  
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In order to solve this problem, we used the Rev protein derived from HIV-1 which binds to and exports RNA containing the cis-acting RRE sequence (Rev Response Element). By fusing the RRE sequence to an RNA of interest and co-expressing Rev protein, complex RNA is exported.
 
  
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===Characterization===
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==Characterization==
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In order to demonstrate the function of the Rev protein and RRE, we fused an RRE [1] to U6 snRNA, which has a complex structure and is not transported by the TAP / p15 pathway. In Xenopus oocytes, we ascertained whether this RNA was exported from the nucleus in a Rev-dependent manner.
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Our team observed that yeast fluoresces EGFP as shown in fig.1. We cultured nematodes using this green fluorescent yeast as a bait. A few days later, we collected and observed the nematodes with a confocal microscope. <br>
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As shown in the figure, the U6-RRE RNA itself remains in the nucleus even 60 minutes after injection. On the other hand, the same RNA was exported to the cytoplasm when Rev was co-injected. From this data, we showed that a combination of RRE and Rev allowed target RNAs to be exported to the cytoplasm.
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It was confirmed that there were individuals emitting strong green fluorescence, and it was possible to confirm for the first time in the world that this nematode is eating <i>S. cerevisiae</i> (fig 2). This fluorescence illuminates the entire intestinal tract of <i>B. xylophilus</i>, and it is shown that this yeast is also useful as a tool for seeing the intestinal structure of nematodes.
  
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<!-- (以下Rev アフリカツメガエルinjection result、and analysis fig) -->
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<html>
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<figure>
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<img src="https://static.igem.org/mediawiki/2017/4/48/GFP_green.jpg" alt="Yeast expressing EGFP" style="float:center;width:350px;height:250px;">
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        </figure>
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Figure1: Observation of <i>S. cerevisiae</i> expressing EGFP from this TDH3 promoter with a fluorescence microscope.
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<br>
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<figure>
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<img src="https://static.igem.org/mediawiki/parts/8/82/B._xEGFP.jpg" alt="<i>B. xylophilus</i> filled with EGFP in its intestine" style="float:center;width:350px;height:250px;">
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        </figure>
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Figure2 : <i>B. xylophilus</i> full of EGFP in its intestine.
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</html>
  
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===Reference===
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According to the above results, this new part makes sufficiently high intensity EGFP expressed in yeast, and the amount is sufficient to observe the intestinal structure of the nematode feeding the yeast. Such usage greatly extends the use applications of BBa_K530008 and BBa_K1875003, making it possible to conduct similar experiments on other nematode species with stylets that prey on yeast.
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[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041468/pdf/gku304.pdf
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==Reference==
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'''[https://parts.igem.org/Part:BBa_K530008 BBa_K530008]''' <br>
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'''[https://parts.igem.org/Part:BBa_K1875003  BBa_K1875003]'''
  
 
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Latest revision as of 02:49, 2 November 2017


TDH3 promoter>EGFP protein

We expressed this gene in Saccharomyces cerevisiae and feed it to B.xylophilus.


Usage and Biology

Aiming for feeding RNAi of B. xylophilus, our team had to confirm that B. xylophilus surely eats S. cerevisiae. To confirm this, we expressed high levels of EGFP in S. cerevisiae by combining BBa_K530008(TDH3 promoter) and BBa_K1875003 (EGFP) and creating BBa_K2403003. Since this part produces a very strong green fluorescence, it is useful for tracking yeast.


Characterization

Our team observed that yeast fluoresces EGFP as shown in fig.1. We cultured nematodes using this green fluorescent yeast as a bait. A few days later, we collected and observed the nematodes with a confocal microscope.
It was confirmed that there were individuals emitting strong green fluorescence, and it was possible to confirm for the first time in the world that this nematode is eating S. cerevisiae (fig 2). This fluorescence illuminates the entire intestinal tract of B. xylophilus, and it is shown that this yeast is also useful as a tool for seeing the intestinal structure of nematodes.

Yeast expressing EGFP
Figure1: Observation of S. cerevisiae expressing EGFP from this TDH3 promoter with a fluorescence microscope.
<i>B. xylophilus</i> filled with EGFP in its intestine
Figure2 : B. xylophilus full of EGFP in its intestine.

According to the above results, this new part makes sufficiently high intensity EGFP expressed in yeast, and the amount is sufficient to observe the intestinal structure of the nematode feeding the yeast. Such usage greatly extends the use applications of BBa_K530008 and BBa_K1875003, making it possible to conduct similar experiments on other nematode species with stylets that prey on yeast.

Reference

BBa_K530008
BBa_K1875003

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]