Part:BBa_K5038044

PT7-LIRA_H01-linker-lacZ-T7TE

Usage and Biology

H01 is a type of ribocomputing system that utilizes a loop-initiated RNA activator (LIRA) motif. This motif binds to an input RNA strand via extended loop domains, which will expose downstream functional domains for subsequent reactions.

Since the loop of H01 can bind to RNA strands, we can modify the sequence on the loop to detect specific RNAs.

1.In the absence of a target RNA, the system adopts the structure shown in Figure 1. This structure features a loop and a stem. The loop contains a sequence complementary to the target RNA strand, and the stem houses the ribosome binding site (RBS) and the initiation codon AUG. Both the RBS and AUG are closed due to the pairing within the stem, thereby blocking translation.

(Figure 1. Schematic of structure 1, LIRA_H01 without input RNA strand)

2.When the target RNA is present, H01 undergoes a conformational change as shown in Figure 2, leading to the formation of the structure shown in Figure 3. In this configuration, the hairpin structure opens, exposing the RBS and AUG. This allows the ribosome to bind to the RBS and start translation at AUG.

(Figure 2. Changes on H01)

(Figure 3. Schematic of structure 2, LIRA_H01 with input RNA strand)

3.A reporter gene is located downstream of H01. By comparing the expression of the reporter gene before and after the loop opens, we can infer the opening of hairpin structure, thus we could detect the target RNA.

Characterization

In this part, we used LacZ as the reporter gene and input01 as the target RNA, and inferred the opening of the hairpin through colour of the solution and OD562 value. This reaction took place in cell-free system

(Figure 2. Colour reaction and OD562 measurement)

As can be seen from Fig.4, when input01 doesn't exist, the CPRG solution is yellow and has a low OD562 value. If input01 is present, the CPRG will convert to CPR and yield a purple solution, which has a high value of OD562. β-gal is the positive control. The results indicates that the LacZ has functioned as a reporter gene successfully, and H01 is an efficient LIRA to detect input01.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 3147
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 3147
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3147
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 3147
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 3147
  
• 1000
  COMPATIBLE WITH RFC[1000]