Difference between revisions of "Part:BBa K1859026"
 
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<p>
 
<p>
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His-RFPの配列を含むmRNAを環状化させる効率を上げるために、私たちはこのジェネレーター[K1859026]をデザインした。
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We designed this generator [BBa_K1859026] to improve circular efficiency of mRNA including His-RFP sequence.
 
</p>
 
</p>
  
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and the 3´side of the intron
 
and the 3´side of the intron
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332005" > [BBa_K1332005] </a>
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332005" > [BBa_K1332005] </a>
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  , and made BBa_K1859015 and BBa_K1859016.
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  , and made
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859015" > [BBa_K1859015] </a>
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and
 +
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859016" > [BBa_K1859016] </a>
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.<br>
 +
 
 +
This part is involved in the second step of the mechanism of the group I intron circularization.<br>
 +
Two exons are connected with each other in the circularization system; furthermore an exon can theoretically be circularized by the system. (shown below)<br>
 +
<img src="https://static.igem.org/mediawiki/2014/a/a1/PIEGIFU3.png" width="650px"></img><br><br>
 +
<img src="https://static.igem.org/mediawiki/2014/d/d2/PIEGIFU2.png" width="600px"></img><br>
 +
 
 +
<b>Fig. about circularization</b><br><br><br>
  
 
Circular parts of iGEM2014 were cloned only sequence of ribozyme involved the splicing from td intron in T4 phage.
 
Circular parts of iGEM2014 were cloned only sequence of ribozyme involved the splicing from td intron in T4 phage.
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However, BBa_K1859015 and BBa_K1859016 were cloned the sequence except the ribozyme extra in addition to
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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.<br>
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332003" > [BBa_K1332003] </a>
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−
and
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332005" > [BBa_K1332005] </a>
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−
.
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</p>
 
</p>
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<center><img src="https://static.igem.org/mediawiki/2015/b/b9/Gifu_outside_1.png" width="380" height="" vspace="25" hspace="" align=""></img></center>
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<p>
 
<p>
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The generator consists of 相補的になるように改良したCircular parts (
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The generator is comprised of Circular parts improved to become complementary (
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859015" > [BBa_K1859015] </a>
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859015" > [BBa_K1859015] </a>
 
  and  
 
  and  
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859016" > [BBa_K1859016] </a>
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859016" > [BBa_K1859016] </a>
 
), His-RFP without stop codon
 
), His-RFP without stop codon
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_BBa_K1332002" >[BBa_K1332002] </a>
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332002" >[BBa_K1332002] </a>
 
, Lacl
 
, Lacl
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_R0010] </a>
 
<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_R0010] </a>
 
, RBS
 
, RBS
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0034" >[BBa_B0034] </a>
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0034" >[BBa_B0034] </a>
 
  and DT
 
  and DT
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_R0010" >[BBa_B0015] </a>
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_B0015" >[BBa_B0015] </a>
 
.
 
.
 
</p>
 
</p>
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 +
<br clear="all">
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<b>About the complementary sequences at outside of both splicing sites</b><br>
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&nbsp;&nbsp;  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>
 +
&nbsp;&nbsp; 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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</br>
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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>
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<center><b>Fig2. Replacing the splicing site and parts constructed</b></center>
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<br>
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&nbsp;&nbsp; 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.
  
 
<p>
 
<p>
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このジェネレーター[BBa_1859026]で作成したRNAは環状mRNAの外側が相補的に結合するように設計されている。
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The RNA made by this generator [BBa_K1859026] was designed to bind outside of circular mRNA complementary.
 
</p>
 
</p>
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 +
<p class="center">
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<img src="https://static.igem.org/mediawiki/2015/1/17/Bba1859026.png" width="800" height="236" vspace="10" hspace="40"></img></p>
  
  
 
<p>
 
<p>
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iGEM2014のジェネレーター[BBa_1332011]で作成した環状mRNAの量、[BBa_1859026]で作成した環状mRNAの量、[BBa_1859027]で作成した環状mRNAの量、[BBa_1859028]で作成した環状mRNAの量を比較すると、環状化の効率は[BBa_1859026]が最も良かった。
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We found that circular efficiency of [BBa_K1859026] was the best,  when we compared with the amount of circular mRNA made by these generators
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1332011" > [BBa_K1332011]</a>
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,
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859024" > [BBa_K1859024]</a>
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,
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<a href= "https://parts.igem.org/wiki/index.php?title=Part:BBa_K1859025" > [BBa_K1859025]</a>
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,[BBa_K1859026].
 
</p>
 
</p>
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</html>
 
</html>

Latest revision as of 02:56, 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] .
This part is involved in the second step of the mechanism of the group I intron circularization.
Two exons are connected with each other in the circularization system; furthermore an exon can theoretically be circularized by the system. (shown below)



Fig. about circularization


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.

Fig2. Replacing the splicing site and parts constructed

   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].