Part:BBa_K5038021
PT7-lacO-LIRA_H01-linker-EGFP-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, while 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 allowing us to 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_H01 produces a large difference between the two conditions of IPTG presence/absence.
Compared to BBa_K5038019, this device has an additional lac operator downstream of T7 promoter, which is the binding site of LacI repression protein and could further prevent the transcription leakage.
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.
| No. | LIRA_H01 plasmids | corresponding input plasmids |
|---|---|---|
| 1 | pET-15b-lacO-H01-linker-EGFP | pCDFDuet-lacO-input01 |
| 2 | pET-15b-lacO-H01-linker-EGFP | pCOLADuet-lacO-input01 |
Considered the BBa_K5038019 has produced the positive result, the verification of single-input LIRA is completed. Therefore, we did not perform complete experiments on two plasmid groups in Table 1.
(Figure 4. Nucleic acid electrophoresis results after co-transformation of plasmids group No.2 in Table 1)
Agarose gel concentration in Figuure 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_H01 itself, we set two controls for each fluorescence results after co-transformation: i.e. IPTG+/- control and input01 presence/absence control.
(Figure 5. Fluorescence mensuration results of plasmids group No.2 in Table 1)
*** indicates p<0.001
This results indicate that the combination of H01 expressed by pET-15b-lacO-H01-linker-EGFP and its corresponding input expressed by pCOLADuet-lacO-input01 plasmid has high sensitivity, but it still has non-negligible fluorescence leakage when the input isn't present.
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]
| None |