Difference between revisions of "Part:BBa K2403003"

Revision as of 10:01, 1 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

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. 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.

Reference

[1]https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041468/pdf/gku304.pdf

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]

@@ Line 7: / Line 7: @@
 <!-- Add more about the biology of this part here -->
-===Usage and Biology===
+==Usage and Biology==
-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.
+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 BBa_K2403003. Since this part produces a very strong green fluorescence, it is useful for tracking yeast.
-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.
-===Characterization===
+==Characterization==
 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.
 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.