Part:BBa_K1859016
the 5´side of the intron[BBa_K1332003] +complementary seqence
[BBa_K1859016] can let mRNA circularize efficiently in E. coli by combining it with [BBa_K1859015] .
Circular Parts, the 3'side of the intron [BBa_K1332005] and the 5'side of the intron [BBa_K1332003] , is what use sequence subcloned ribozyme involved self‐splicing in T4 Phage. By associating Circular Parts on both ends of optional sequence, you can circularize mRNA making its DNA.
As for details of circular mRNA, refer to ‘iGEM Gifu 2014' .
We improved the 5´side of the intron [BBa_K1332003] of iGEM2014 and made [BBa_K1859016]. Circular parts of iGEM2014 are cloned only sequence of ribozyme involved the splicing from td intron in T4 phage. However, [BBa_K1859016] is cloned the sequence except the ribozyme extra in addition to [BBa_K1332003] .
We attached this part[BBa_K1859016] to 3'sides of His-RFP without stop codon [BBa_K1332002] . Then, it was connected to 3'sides of [BBa_K1859015] . In addition,Lacl [BBa_R0010] , RBS [BBa_B0034] and DT [BBa_B0015] was conbined with it, making [BBa_K1859026] . [BBa_K1859026] is the generator to improve circular efficiency of mRNA. The sequence that cloned extra in [BBa_K1859016] has complementarity with the sequence that cloned extra in [BBa_K1859015].
You can let mRNA circularize efficiently by using [BBa_K1859026] ([BBa_K1859016] and [BBa_K1859015] ) than using the generator of 2014 [BBa_K1332011] ( [BBa_K1332005] and [BBa_K1332003] ).
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.
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].
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