Part:BBa_K5115071
NixA, without stop codon
Introduction
NixA is a high-affinity nickel transporter in helicobacter pylori. It belongs to the NiCoT family of transporters and facilitates the import of Ni²⁺ ions across the bacterial cytoplasmic membrane. These nickel ions make up the active sites of urease and hydrogenase, enzymes crucial for H. pylori's ability to survive in the highly acidic environment of the stomach.[1] In our design, the NixA is expressed on the cell membrane of E.coli and undertake the task of importing nickel into the bacteria.
Usage and Biology
We delete the stop codon of this part to link it with BBa_K5115085(F1v).For more information about their experiment, please check BBa_K5115086(NixA-F1v).
Characterization
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| Figure 1. The alphafold structure of NixA.[2]
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| Figure 2. Comparison of Ni²⁺ Uptake Efficiency by Different ‘’E. coli’’ in 20 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ absorbed by ‘’E. coli’’ expressing different constructs after 5 hours of growth in a medium containing 20 mg/L Ni²⁺ (‘’E. coli’’ strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 20 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0306, Dunnett’s post-test). |
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| Figure 3. Comparison of Ni²⁺ Uptake Efficiency by Different ‘’E. coli’’ in 30 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ absorbed by ‘’E. coli’’ expressing different constructs after 5 hours of growth in a medium containing 30 mg/L Ni²⁺ (‘’E. coli’’ strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 30 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0052, Dunnett’s post-test). |
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| Figure 4. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 50 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ absorbed by ‘’E. coli’’ expressing different constructs after 5 hours of growth in a medium containing 50 mg/L Ni²⁺ (‘’E. coli ‘’strain: BL21 DE3, induced with 1 mM IPTG). Ni²⁺ uptake was calculated based on the difference between initial and final concentrations in the supernatant, divided by 50 mg/L. The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. Culture for 5 hours, at 37°C with a rotating speed at 220 rpm. Regarding NixA-F1v and F1v-NixA, AP20187 is a synthetic dimerizer that can be used to induce homodimerization of F1v domain. Three biological replicates were performed for each condition, and error bars represent the standard errors of the means (SEM) of these replicates. ANOVA test shows that all constructs increase Ni²⁺ uptake significantly compared to the control. Bacteria expressing NixA-F1v exhibit the highest Ni²⁺ uptake efficiency (p = 0.0020, Dunnett’s post-test). |
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]
References
- ↑ Fischer, F., Robbe-Saule, M., Turlin, E., Mancuso, F., Michel, V., Richaud, P., Veyrier, F. J., Reuse, H. D., & Vinella, D. (2016). Characterization in Helicobacter pylori of a Nickel Transporter Essential for Colonization That Was Acquired during Evolution by Gastric Helicobacter Species. PLOS Pathogens, 12(12), e1006018.
- ↑ Abramson, J., Adler, J., Dunger, J., Evans, R., Green, T., Pritzel, A., Ronneberger, O., Willmore, L., Ballard, A. J., Bambrick, J., Bodenstein, S. W., Evans, D. A., Hung, C.-C., O’Neill, M., Reiman, D., Tunyasuvunakool, K., Wu, Z., Žemgulytė, A., Arvaniti, E., … Jumper, J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500.
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