Part:BBa_K5411003
GlnRN79-LeucinzipperA3.5-GlnRC40
LeucinzipperA3.5 (BBa_K5411020) is fused inside GlnR(BBa_K5411002).
Usage and Biology
GlnR is a transcription factor in Bacillus subtilis. When Bacillus subtilis's glutamine synthetase, GlnA, is feedback-inhibited by its product, glutamine, GlnR gains the ability to bind to specific promoters [1]. GlnR binds to particular DNA sequences, such as the GlnRA promoter, as a dimer. However, due to a self-inhibitory domain located near its C-terminus, GlnR normally cannot dimerize or bind to DNA. In the presence of GlnA-FBI (feedback-inhibited GlnA), this self-inhibitory domain binds to GlnA, neutralizing its inhibitory effect and allowing GlnR to bind to DNA [2][3]. Since GlnA-FBI facilitates GlnR's binding to DNA in a glutamine-dependent manner, GlnR acts as a repressor in Bacillus subtilis's nitrogen response system.
The mechanism involving GlnR has already been applied in an in vivo ammonium biosensor [4]. Inspired by this, Kyoto 2024 designed a cell-free biosensor.
Specifically, GlnR and GlnA are located around the DNA when ammonia concentration increases. By fusing LeucinzipperAN3.5 to GlnR and GlnA, and fusing LeucinzipperBN3.5 to T7RNAP, the interaction between the leucine zippers can induce the recruitment of T7RNAP around the promoter only when ammonia concentration is high and GlnR is bound to the DNA. By using the low-affinity T7Promoter d1 (BBa_K5411028[5]) instead of the regular T7 promoter, transcription can be initiated exclusively through this recruitment mechanism.
Design and modeling
We fused GlnR with a leucine-zipper structure, aiming to add the functionality of the leucine-zipper without affecting the original function of GlnR. Two potential fusion sites were considered: the N-terminus and the linker sequence between Helix 4 and Helix 5. The selected design involved inserting LeucinzipperA3.5 into the linker region.
Using Alphafold3 [6] for modeling, we aimed to design a structure that would not disrupt GlnR’s function. The resulting structure successfully prevented dimerization through interaction with GlnR itself, while positioning the C-terminus, crucial for interaction with GlnA-FBI, in an appropriate orientation to interact with GlnR. The linker used was (GGGGS)5. When compared to the crystal structure of GlnR, the model showed a low RMSD value of 0.757, indicating minimal structural disruption.
Purification
The plasmid was constructed by introducing synthetic DNA purchased from IDT into the pET15b 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 | Elution Buffer |
|---|---|---|
| 50mM Tris-HCl (pH8.0) | 50mM Tris-HCl (pH8.0) | 50mM Tris-HCl (pH8.0) |
| 300mM NaCl | 300mM NaCl | 300mM NaCl |
| 5mM MgCl2 | 5mM MgCl2 | 5mM MgCl2 |
| 5% v/v Glycerol | 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
References
[1]Fisher SH, Wray LV. Bacillus subtilis glutamine synthetase regulates its own synthesis by acting as a chaperone to stabilize GlnR–DNA complexes. Proceedings of the National Academy of Sciences. 2008;105(3):1014-1019. doi:https://doi.org/10.1073/pnas.0709949105
[2]Wray LV, Fisher SH. Bacillus subtilis GlnR contains an autoinhibitory C-terminal domain required for the interaction with glutamine synthetase. Molecular Microbiology. 2008;68(2):277-285. doi:https://doi.org/10.1111/j.1365-2958.2008.06162.x
[3]Travis BA, Peck JV, Salinas R, et al. Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria. Nature Communications. 2022;13(1). doi:https://doi.org/10.1038/s41467-022-31573-0
[4]Xiao Y, Jiang W, Zhang F. Developing a Genetically Encoded, Cross-Species Biosensor for Detecting Ammonium and Regulating Biosynthesis of Cyanophycin. ACS Synthetic Biology. 2017;6(10):1807-1815. doi:https://doi.org/10.1021/acssynbio.7b00069
[5]BBa_K5411028
[6]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]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |