Difference between revisions of "Part:BBa K1859026"
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| + | <b>About the complementary sequences at outside of both splicing sites</b><br> | ||
| + | There are the complementary sequences inside the splicing site in this intron. We thought that the sequence brings one splicing site close to the other one and ensures the reaction takes place.<br> | ||
| + | Therefore, we cloned splicing sites with complementary sequences in intron and made it into parts. Moreover, we made the device which express circular mRNA by using this part, and made its express in <i>E. coli</i>. Then, complementary sequences are included in outside of both splicing sites in RNA. | ||
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| + | <p class="center"><img src="https://static.igem.org/mediawiki/2015/e/e1/About-at-autside.png" width="427" height="277" vspace="25" hspace="50"></img></p> | ||
| + | <center><b>Fig2. Replacing the splicing site and parts constructed</b></center> | ||
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| + | We used <a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859026"style="text-decoration:none;" >BBa_K1859026</a> for generator of outside complementarity. | ||
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Revision as of 02:20, 25 September 2015
mRNA circularize efficiently generator (outside complementarity)
We designed this generator [BBa_K1859026] to improve circular efficiency of mRNA including His-RFP sequence.
First, we improved Circular Parts of iGEM2014,the 5´side of the intron
[BBa_K1332003]
and the 3´side of the intron
[BBa_K1332005]
, and made
[BBa_K1859015]
and
[BBa_K1859016]
.
Circular parts of iGEM2014 were cloned only sequence of ribozyme involved the splicing from td intron in T4 phage.
However, [BBa_K1859015] and [BBa_K1859016] were cloned the sequence extra in addition to the ribozyme. The secondary structure as the mRNA is shown below.
The generator is comprised of Circular parts improved to become complementary ( [BBa_K1859015] and [BBa_K1859016] ), His-RFP without stop codon [BBa_K1332002] , Lacl [BBa_R0010] , RBS [BBa_B0034] and DT [BBa_B0015] .
About the complementary sequences at outside of both splicing sites
There are the complementary sequences inside the splicing site in this intron. We thought that the sequence brings one splicing site close to the other one and ensures the reaction takes place.
Therefore, we cloned splicing sites with complementary sequences in intron and made it into parts. Moreover, we made the device which express circular mRNA by using this part, and made its express in E. coli. Then, complementary sequences are included in outside of both splicing sites in RNA.
We used BBa_K1859026 for generator of outside complementarity.
The RNA made by this generator [BBa_K1859026] was designed to bind outside of circular mRNA complementary.
We found that circular efficiency of [BBa_K1859026] was the best, when we compared with the amount of circular mRNA made by these generators [BBa_K1332011] , [BBa_K1859024] , [BBa_K1859025] ,[BBa_K1859026].