Part:BBa_K5038036

PT7-LIRA_ANDgate2-linker-EGFP-T7TE

Usage and Biology

LIRA_ANDgate2 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_ANDgate2 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_ANDgate2 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_ANDgate2 achieves the "AND" detection of two miRNAs through this LIRA structure.

(Figure 3. MiRNA-B binds to LIRA_ANDgate2-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_ANDgate2 and the corresponding input plasmids into E.coli BL21(DE3). Plasmid pCOLADuet-1 contained the device of PT7-LIRA_ANDgate2-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-transformation)

As can be seen from Fig.4, plasmids are all digested by HpaI and produced the correct bands marked as a 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_ANDgate2)

As can be seen from Fig.5, when inputRNAs do not exist, LIRA_ANDgate2 occurs a low fluorescence leakage. The fluorescence produced when only input142 is present is essentially the same intensity as the fluorescence produced when both inputs are present. This results indicates that LIRA_ANDgate2 does not implement the function of an AND gate.

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]