Part:BBa_K5115085
F1v (FKBP with F36V mutation) without stop codon
Introduction
This part is FKBP with F36V mutation, which is a signal transducer. It's used to bind with the target protein. 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.[1]
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| 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.
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| Figure 2. While using our iDimerize kit, the explanation of AP20187 in the kit's User Manual.
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Usage and Biology
In our experiment, we adopted the iDimerize kit. To learn fully about this kit, please check iDimerize™ Inducible Homodimer System User Manual.
For convenient connections between the Fiv and NixA, we delete the stop codon of F1v. We link this part with BBa_K5115071(NixA) in two different ways to find the more efficient way of dimerizing the NixA protein. It turns out that linking F1v with the C end of NixA is a better choice, please check BBa_K5115086(NixA-F1v)for more details about our experiment.
Characterization
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| Figure 1. 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 2. 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 3. 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
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
- ↑ 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.
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