Part:BBa_K5038032

PT7-LIRA_H210_10/12-linker-EGFP-T7TE

Usage and Biology

LIRA_H210_10/12 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.

"10/12" indicates that this LIRA system has a stem-loop ratio of 10/12 when bound to the corresponding input, i.e., in the input sequence, 10 bases are bound to the stem of the LIRA hairpin and 12 bases are bound to the loop.

(Figure 1. Schematic of LIRA_H210_10/12)

Blue: miRNA binding site, miR-210-3p binds to this area
Purple: RBS
Yellow: AUG, initiation codon

Characterization

Firstly, we co-transformed LIRA_H210_10/12 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_H210_10/12-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, Lane3, Lane7 and Lane11 are the plasmids related to BBa_K5038032, 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_10/12)

Fig.3 indicates that LIRA_H210_10/12 has poor performance in detecting input210. When input210 exists, the elevation of the fluorescence intensity of LIRA_H210_10/12 is not significant. The results illustrates that LIRA_H210_10/12 cannot detect input210 efficiently. Based on these results, we don't recommend H210 with a stem loop ratio of 10/12 as input210 detector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]