Part:BBa_K5038027
PT7-LIRA_H142_3/20-linker-EGFP-T7TE
Usage and Biology
LIRA_H142_3/20 is a LIRA system which could specifically detect hsa-miR-142-3p, and has the same detection principle to LIRA_H01. There is a complementary sequence of hsa-miR-142-3p 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.
"3/20" indicates that this LIRA system has a stem-loop ratio of 3/20 when bound to the corresponding input, i.e., in the input sequence, 3 bases are bound to the stem of the LIRA hairpin and 20 bases are bound to the loop.
(Figure 1. Schematic of LIRA_H142_3/20)
Blue: miRNA binding site, miR-142-3p binds to this area
Purple: RBS
Yellow: AUG, initiation codon
Characterization
Firstly, we co-transformed LIRA_H142_3/20 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_H142_3/20-linker-EGFP-T7TE, and plasmid pET-15b and pCDFDuet-1 contained the device of PT7-LIRA_input142-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_K5038027, and are all digested by HpaI. The correct bands are marked as a red star. The results indicates that the co-transformation is successful.
In this part, we used EGFP as the reporter gene and input142 as the target RNA, and inferred the opening of the hairpin through the fluorescence intensity.
(Figure 3. EGFP fluorescence of LIRA_H142_3/20)
Fig.3 indicates that LIRA_H142_3/20 has poor performance in detecting input142. When input142 exists, the elevation of the fluorescence intensity of LIRA_H142_3/20 is not significant. The results illustrates that LIRA_H142_3/20 cannot detect input142 efficiently. Based on these results, we don't recommend H142 with a stem loop ratio of 3/20 as input142 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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