Part:BBa_K5411010
MS2 Coat Protein-C-T7RNAP
MS2 coat protein (BBa_K5411024) and C-T7RNAP (BBa_K5411023) are fused and N-his tagged.
Usage and Biology
Split NT7 is a split T7 RNA polymerase (1-179) that was engineered using directed evolution in previous studies [1] to reduce its spontaneous reassembly, allowing it to be reconstituted primarily through proximity induced by protein-protein interactions. This feature makes it useful in systems that trigger transcription via such interactions.
NasR is a protein derived from Klebsiella oxytoca. It contains an ANTAR domain, an RNA-binding domain, which, in vivo, binds to terminator RNA sequences in the presence of nitrate, thereby preventing transcription termination [2]. Upon binding to nitrate, NasR undergoes a conformational change that enables it to bind to specific RNA sequences. This mechanism has already been exploited for the development of FRET-based biosensors [3].
Kyoto 2024 designed a system leveraging NasR's ability to bind RNA in the presence of nitrate. By incubating N-T7RNAP-NasR (BBa_K5411009) alongside this part, Split T7RNAP is reconstituted on the RNA in the presence of nitrate, thereby promoting transcription.
Design and modeling
The following diagram illustrates the arrangement of proteins envisioned for the reconstitution of Split T7RNAP on RNA. In the diagram, green protein represents NasR, light blue one is C-T7RNAP, red one is N-T7RNAP, and yellow one is MCP. The predicted structures were generated using Alphafold3 [4]. If this arrangement can be replicated on RNA, it was hypothesized that the reconstitution of T7RNAP can be achieved. Given that NasR is larger than MS2, a GGGGS linker is placed between NasR and N-Split T7RNAP, and a (GGGGS)5 linker is placed between MS2 and C-Split T7RNAP.
Purification
The plasmid was constructed by introducing synthetic DNA purchased from IDT into the pET11a vector using XE cocktail assembly. The sequence was then verified to match the target sequence through Sanger sequencing.
The designed plasmid was transformed into BL21 competent cells and cultured overnight O/N on LB agar plates containing 50 mg/L ampicillin. The resulting colonies were suspended and subsequently cultured in LB Broth supplemented with 50 mg/L ampicillin. Initially, the culture was incubated at 37°C and 160 rpm in a shaker incubator, with OD monitored. When the OD reached 0.6, IPTG was added to a final concentration of 0.5mM to induce protein expression. The culture was then incubated overnight at 15°C and 160 rpm. After culturing, the bacterial cells were pelleted by centrifugation and resuspended in Lysis Buffer (Table). The cells were then disrupted by sonication, and after centrifugation to remove cell debris, the supernatant was mixed with pre-washed Ni-NTA beads. The beads were washed three times with 25 mL of Wash Buffer, followed by loading the suspension onto a protein purification column and performing four additional washes with 6 mL of buffer each time. Finally, the protein was eluted in six fractions using Elution Buffer adjusted to imidazole concentrations of 50 mM, 100 mM, 200 mM, and 300 mM.
| Lysis Buffer | Wash Buffer 1 | Wash Buffer 2 | Elution Buffer |
|---|---|---|---|
| 20mM Tris-HCl (pH 7.8) | 20mM Tris-HCl (pH 7.8) | 40mM Tris-HCl (pH 7.8) | 40mM Tris-HCl (pH 7.8) |
| 500mM NaCl | 500mM NaCl | 200mM NaCl | 200mM NaCl |
| 30mM Imidazole | 30mM Imidazole | 50, 100, 200, 300mM Imidazole |
The eluted fractions obtained from protein purification were subjected to SDS-PAGE, and the following bands were observed on the gel.
Figure 1 | The result of SDS-PAGE
However, despite multiple purification attempts while adjusting mass culture and purification conditions as detailed in the notes, clear SDS-PAGE bands were not observed at the expected positions. This is likely due to decreased solubility and folding issues caused by the split design. Given that other composite parts containing split proteins, such as NasR-NT7 and NT7-NLP7-CT7, have been successfully purified to some extent, it suggests that purification might be achievable through fusion with a solubility tag or by using an expression system other than the pET system. This represents a milestone that could facilitate more efficient research in our future studies and in other applications of split proteins in vitro.
References
[1]Pu J, Zinkus-Boltz J, Dickinson BC. Evolution of a split RNA polymerase as a versatile biosensor platform. Nature Chemical Biology. 2017;13(4):432-438. doi:https://doi.org/10.1038/nchembio.2299
[2]Goodson JR, Zhang C, Trettel DS, et al. An autoinhibitory mechanism controls RNA‐binding activity of the nitrate‐sensing protein NasR. Molecular Microbiology. 2020;114(2):348-360. doi:https://doi.org/10.1111/mmi.14517
[3]Chen YN, Cartwright HN, Ho CH. In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor. Science Advances. 2022;8(42). doi:https://doi.org/10.1126/sciadv.abq4915
[4]Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:1-3. doi:https://doi.org/10.1038/s41586-024-07487-w
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2550
Illegal AgeI site found at 1762 - 1000COMPATIBLE WITH RFC[1000]
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