Difference between revisions of "Part:BBa K5115086"
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===Introduction=== | ===Introduction=== | ||
| − | This part | + | This part combines [https://parts.igem.org/Part:BBa_K5115071 BBa_K5115071(NixA)]with [https://parts.igem.org/Part:BBa_K5115085 BBa_K5115085(F1v)]. |
| + | |||
| + | 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<ref>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.</ref>. F1v is a signal transducer, binding NixA at its C end. The working procedure of F1v need the use of AP20187, which is a small molecule inducers. The addition of AP20187 to live cells expressing a F1v-tagged fusion protein induces self-association of the fusion protein by promoting the interaction of the dimerization domains.<ref>Clackson, T., Yang, W., Rozamus, L. W., Hatada, M., Amara, J. F., Rollins, C. T., Stevenson, L. F., Magari, S. R., Wood, S. A., Courage, N. L., Lu, X., Cerasoli, F., Gilman, M., & Holt, D. A. (1998). Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10437–10442.</ref> | ||
| + | |||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/registry/f1v.jpg" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''Figure 1. The working procedure of AP20187 and F1v according to the iDimerize™ Inducible Homodimer System User Manual. The F1v is combined with the target protein, which is NixA in our experiment, and the AP20187 can induce self-association of the NixA. | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | |||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/registry/ap20187-in-the-manual.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''Figure 2. While using our iDimerize kit, the explanation of AP20187 in the kit's User Manual. | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | |||
| + | ===Usage and Biology=== | ||
| + | This part is built to introduce NixA into the ''E.coli'' and enhance its transporting abilities. We combined all of our parts concerning Nickel ions' transporting and detoxifying into [https://parts.igem.org/Part:BBa_K5115068 BBa_K5115068(mineral nickel module)], please visit this part to learn more details about our design and experiments. | ||
===Characterization=== | ===Characterization=== | ||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/registry/nixa-f1v.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''Figure 1. The alphafold structure of NixA-F1v.<ref> 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. </ref> | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | |||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/20-mg-l-single-plasmid.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''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). | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | |||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/30-mg-l-single-plasmid.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''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). | ||
| + | ''' | ||
| + | |||
| + | |} | ||
| + | |||
| + | {| | ||
| + | | <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/ni-results/50-mg-l-single-plasmid.png" alt="contributed by Fudan iGEM 2024"></html> | ||
| + | |- | ||
| + | | '''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). | ||
| + | ''' | ||
| + | |||
| + | |} | ||
Latest revision as of 12:03, 2 October 2024
NixA-F1v
Introduction
This part combines BBa_K5115071(NixA)with BBa_K5115085(F1v).
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[1]. F1v is a signal transducer, binding NixA at its C end. The working procedure of F1v need the use of AP20187, which is a small molecule inducers. The addition of AP20187 to live cells expressing a F1v-tagged fusion protein induces self-association of the fusion protein by promoting the interaction of the dimerization domains.[2]
|
| Figure 1. The working procedure of AP20187 and F1v according to the iDimerize™ Inducible Homodimer System User Manual. The F1v is combined with the target protein, which is NixA in our experiment, and the AP20187 can induce self-association of the NixA.
|
|
| Figure 2. While using our iDimerize kit, the explanation of AP20187 in the kit's User Manual.
|
Usage and Biology
This part is built to introduce NixA into the E.coli and enhance its transporting abilities. We combined all of our parts concerning Nickel ions' transporting and detoxifying into BBa_K5115068(mineral nickel module), please visit this part to learn more details about our design and experiments.
Characterization
|
| Figure 1. The alphafold structure of NixA-F1v.[3]
|
|
| 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). |
|
| 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). |
|
| 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.
- ↑ Clackson, T., Yang, W., Rozamus, L. W., Hatada, M., Amara, J. F., Rollins, C. T., Stevenson, L. F., Magari, S. R., Wood, S. A., Courage, N. L., Lu, X., Cerasoli, F., Gilman, M., & Holt, D. A. (1998). Redesigning an FKBP–ligand interface to generate chemical dimerizers with novel specificity. Proceedings of the National Academy of Sciences of the United States of America, 95(18), 10437–10442.
- ↑ 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.