Part:BBa_K5038040

PT7-LIRA_ANDgate5-linker-lacZ-T7TE

Usage and Biology

LIRA_ANDgate5 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_ANDgate5 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_ANDgate5 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_ANDgate5 achieves the "AND" detection of two miRNAs through this LIRA structure.

(Figure 3. MiRNA-B binds to LIRA_ANDgate5-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 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) , yield a purple solution.

(Figure 4. Color reaction and OD562 measurement of LIRA_ANDgate5)

As shown in Fig. 4, when input RNAs are absent, the CPRG solution in the cell-free system is yellow. When only one input RNA is present, the CPRG solution remains yellow, indicating that the LIRA hairpin did not open to express lacZ. Only when both input RNAs are present does CPRG convert to CPR, causing the solution to turn purple. Based on these results, we conclude that LIRA_ANDgate5 fully utilizes the functionality of the AND gate and accurately detects two miRNAs.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3200
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3200
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3200
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3200
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3200
  
• 1000
  COMPATIBLE WITH RFC[1000]