Part:BBa_K5038033

PT7-LIRA_ANDgate1-linker-EGFP-T7TE

Usage and Biology

LIRA_ANDgate1 is a two-input LIRA system. Unlike LIRA_H01, this system initiates downstream gene expression only when both inputs are present. Without binding to any input RNA, this part presents the conformation shown in Figure 1.

(Figure 1. Conformation of LIRA_ANDgate1 without any input)

When the loop on the short arm binds to the corresponding input RNAs, this system will change its conformation shown in Figure 2.

(Figure 2. LIRA_ANDgate1 binds to miRNA-A)

At this point, if the loop on the long arm binds to its corresponding input RNA, the RBS and AUG will be exposed, thus initiating downstream transcription. When only the input RNA corresponding to the loop on the long arm exists in the system (i.e., only inputB), the hairpin structure will not be opened due to the low instability. LIRA_ANDgate1 achieves the "AND" detection of two miRNAs through this LIRA structure.

(Figure 3. MiRNA-B binds to LIRA_ANDgate1-miRNA-A complex)

In our experiments, the loop on the short arm is used to detect hsa-miR-142-3p, and the loop on the long arm is used to detect hsa-miR-210-3p.

Characterization

Firstly, we co-transformed the LIRA_ANDgate1 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_ANDgate1-linker-EGFP-T7TE, plasmid pET-15b contained PT7_LIRA_input142-T7TE and plasmid pCDFDuet-1 contained PT7-LIRA_input210-T7TE. The results of 1% agarose gel electrophoresis are shown in Fig.4.

(Figure 4. 1% agarose gel electrophoresis results after co-transformtion)

As can be seen from Fig.4, plasmids are all digested by HpaI and produced the correct bands marked as red stars. The results indicates that the co-transformation is successful.

In this part, we used EGFP as the reporter gene and input142 and input210 as the target RNAs, and inferred the opening of the hairpin through the fluorescence intensity.

(Figure 5. EGFP fluorescence of LIRA_ANDgate1)

As can be seen from Fig.5, LIRA_ANDgate1 occurs a significant fluorescence leakage. When inputRNAs do not exist, LIRA_ANDgate1 also unlocks its hairpin to express downstream reporter gene EGFP and produces fluorescence.

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]