Part:BBa_K1859015

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].