Part:BBa_K5038034
PT7-LIRA_ANDgate1-linker-lacZ-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
In this part, we planned to use lacZ as the reporter gene and input142 and input210 as the target RNAs. When lacZ (β-gal) is present, it will convert the yellow‐orange CPRG substrate into the chlorophenol red (CPR) , which will turn into a purple solution. Considering the unsatisfactory results of BBa_K5036033, we did not perform experiments on the LIRA_ANDgate1.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3202
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3202
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3202
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3202
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3202
- 1000COMPATIBLE WITH RFC[1000]
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