Part:BBa_K5038028
PT7-LIRA_H142_8/15-linker-EGFP-T7TE
Usage and Biology
LIRA_H142_8/15 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.
"8/15" indicates that this LIRA system has a stem-loop ratio of 8/15 when bound to the corresponding input, i.e., in the input sequence, 8 bases are bound to the stem of the LIRA hairpin and 15 bases are bound to the loop.
(Figure 1. Schematic of LIRA_H142_8/15)
Blue: miRNA binding site, miR-142-3p binds to this area
Purple: RBS
Yellow: AUG, initiation codon
Characterization
Firstly, we co-transformed LIRA_H142_8/15 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_H142_8/15-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, Lane2, Lane6 and Lane10 are the plasmids related to BBa_K5038028, 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_8/15)
Fig.3 indicates that LIRA_H142_8/15 has poor performance in detecting input142. When input142 does not exist, LIRA_H142_8/15 occurs a significant fluorescence leakage. Based on these results, we don't recommend H142 with a stem loop ratio of 8/15 as input142 detector.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 33
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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