Part:BBa_K5038031
PT7-LIRA_H210_7/15-linker-EGFP-T7TE
Usage and Biology
LIRA_H210_7/15 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.
"7/15" indicates that this LIRA system has a stem-loop ratio of 7/15 when bound to the corresponding input, i.e., in the input sequence, 7 bases are bound to the stem of the LIRA hairpin and 15 bases are bound to the loop.
(Figure 1. Schematic of LIRA_H210_7/15)
Blue: miRNA binding site, miR-210-3p binds to this area
Purple: RBS
Yellow: AUG, initiation codon
Characterization
Firstly, we co-transformed LIRA_H210_7/15 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_H210_7/15-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, Lane2, Lane6 and Lane10 are the plasmids related to BBa_K5038031, 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_7/15)
Fig.3 indicates that the LIRA_H210_7/15 has poor performance in detecting input210. It has a significant fluorescence leakage when input210 doesn’t exist, and produces a lower fluorescence intensity when input210 exists than the leakage. Based on these results, we do not recommend H210 with a stem/loop ratio of 7/15 as input210 detector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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