Part:BBa_K5038030
PT7-LIRA_H210_2/20-linker-EGFP-T7TE
Usage and Biology
LIRA_H210_2/20 is a LIRA system which could specifically detect hsa-miR-210-3p, and has the same detection principle to LIRA_H01. There is a complementary sequence of hsa-miR-210-3p on the loop of the hairpin structure, the RBS and initiation codon AUG will be exposed when the complementary sequence is bound to the target RNA, thereby activates translation.
"2/20" indicates that this LIRA system has a stem-loop ratio of 2/20 when bound to the corresponding input, i.e., in the input sequence, 2 bases are bound to the stem of the LIRA hairpin and 20 bases are bound to the loop.
(Figure 1. Schematic of LIRA_H210_2/20)
Blue: miRNA binding site, miR-210-3p binds to this area
Purple: RBS
Yellow: AUG, initiation codon
Characterization
Firstly, we co-transformed LIRA_H210_2/20 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_H210_2/20-linker-EGFP-T7TE, and plasmid pET-15b and pCDFDuet-1 contained the device of PT7-LIRA_input210-T7TE. The results of 1% agarose gel electrophoresis are shown in Fig.2.
(Figure 2. 1% agarose gel electrophoresis results after co-transformation)
As can be seen from Fig.2, Lane1, Lane5 and Lane9 are the plasmids related to BBa_K5038030, and are all digested by HpaI. The correct bands are marked as a red star. This results indicates that the co-transformation is successful.
In this part, we used EGFP as the reporter gene and input210 as the target RNA, and inferred the opening of the hairpin through the fluorescence intensity.
(Figure 3. EGFP fluorescence of LIRA_H210_2/20)
*** indicates p <0.001
Fig.3 indicates that the LIRA_H210_2/20 could detect the input210 correctly, and LIRA_H210_2/20 has better effect on detecting input210 which was deposited in plasmid pCDFDuet-1.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 46
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 34
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