Part:BBa_K5038020

PT7-LIRA_H05-linker-EGFP-T7TE

Usage and Biology

H05 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 H05 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_H05 without input RNA strand)

2.When the target RNA is present, H05 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 H05)

(Figure 3. Schematic of structure 2, LIRA_H05 with input RNA strand)

3.A reporter gene is located downstream of H05. 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 our experiments, we used EGFP as the reporter gene and input01 as the target RNA, and inferred the opening of the hairpin through the fluorescence intensity.

T7 promoter could be induced by IPTG, so the intracellularly concentration of LIRA_H05 produces a large difference between the two conditions of IPTG presence/absence.

We generated this sequence intracellularly by placing the sequence of this composite part on a specific plasmid, then deposit the switch plasmids and its corresponding input plasmids into E.coli BL21(DE3) through co-transformation.

(Table 1. LIRA_H05 plasmid and the corresponding input plasmids)

No.	LIRA_H05 plasmids	corresponding input plasmids
1	pET-15b-H05-linker-EGFP	pCDFDuet-input05
2	pET-15b-H05-linker-EGFP	pCOLADuet-input05

Considered the two plasmids groups have the same experimental purpose, and the functional verification of single-input LIRA can be completed if one of the groups produces a positive result, we only perform complete experiments (i.e., nucleic acid electrophoresis, fluorescent mensuration, Western Blot, SDS-PAGE) of plasmid group No.1 in Table 1.

(Figure 4. Nucleic acid electrophoresis results after co-transformation of plasmids group No.1 in Table 1)
K5038030: pET-15b-H05-linker-EGFP
K5038034: pCDFDuet-input05

Agarose gel concentration in Figure 4 is 1g Agar/100mL 1x TAE, the kinds of restriction endonucleases are marked in the electrophoresis picture. The concentration of each endonucleases in the system was 1u/μL. As can be seen from Figure 4, the co-transformation experiment of two plasmids was successful.

Centrifuge the bacteria solution and get the deposit after IPTG induction (final concentration = 0.1mM) about 4h, then suspend by PBS, centrifuge again after ultrasonic bacteria disruption, collect protein sample.

Dilute all protein samples to 1.8μg/μL, determine fluorescence intensity at excitation wavelength of 488nm, emission wavelength of 525nm, medium-high PMT gain, the results are shown in Figure 5.

Considering the possible EGFP translation leakage in LIRA_H05 itself, we set two controls for each fluorescence results after co-transformation: i.e. IPTG+/- control and input05 presence/absence control.

(Figure 5. Fluorescence mensuration results of plasmids group No.1 in Table 1)
*** indicates p <0.001