Difference between revisions of "Part:BBa K2908666"
 
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<b><i>References:</i></b><br/>
 
<b><i>References:</i></b><br/>
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[1] Ebert, M.S., and Sharp, P.A. (2010). MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050.<br/>
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<i>[1] Ebert, M.S., and Sharp, P.A. (2010). MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050.<br/>
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[2] Tomas Barta, Lucie Peskova & Ales Hampl, (2018), miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico, Scientific Reports.
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[2] Tomas Barta, Lucie Peskova & Ales Hampl, (2018), miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico, Scientific Reports.</i>

Latest revision as of 19:34, 21 October 2019


miR148b-sponge

This part is designed by miRNASong(an online software) and synthesized by company.It is used to down-regulate the level of miR148b in heterogenous TNBC cells.

T--CSU_CHINA--2908666.jpg

Figure1. Picture A shows the sequence of this part; The heat map shows as (B).


Construction
Firstly, obtain the basic sequence information of miRNA from miRBASE, analyze which miRNA subtype is specifically described according to the literature table provided, obtain the sequence and identify the seed sequence.[1] The miRNAsong website[2] was used to design the basic sponge framework, the free energy cutoff was 25Kcal/mol, and the miss was checked by 2-8seed classic matching. Since more than one BS will be connected in the following experiments, spacer connection is directly designed between two BS in the design stage to facilitate the judgment of the overall change after spacer connection. Then manually annotate the mismatch site, spacer sequence. The website does not consider the off-target effect at all, and most of the results are so severe that the 9-12 base combination needs to be manually changed according to the site. Get the combination with lower miss, perform secondary structure verification, and record the passed combination.

T--CSU_CHINA--Figure5_small.jpg

Figure2. This figure demonstrates that when combining the sponge into a test plasmid, miR148b-sponge can efficiently inhibit the endogenous miR148b compared to miR141-sponge when driven by the TNBC-specific promoter s(ESR1)p.


Usage and Biology -The microRNA (miRNA) “sponge” method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems.
Here we designed the sponge specifically targeting miR48b, in order to make it a switch in the complex system for 2019 CSU_CHINA or for other teams in the future, moreover.


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] Ebert, M.S., and Sharp, P.A. (2010). MicroRNA sponges: progress and possibilities. RNA 16, 2043–2050.
[2] Tomas Barta, Lucie Peskova & Ales Hampl, (2018), miRNAsong: a web-based tool for generation and testing of miRNA sponge constructs in silico, Scientific Reports.