Part:BBa_K5038038
PT7-LIRA_ANDgate2-linker-lacZα-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
In this part, we used lacZα as the reporter gene and input142 and input210 as the target RNAs. LacZα can perform α-complementation with LacZω, then form β-gal and convert the yellow‐orange CPRG substrate into the chlorophenol red (CPR) , which will turn into a purple solution. Considering the unsatisfactory results of BBa_K5036036, we did not perform experiments on the LIRA_ANDgate2.
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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