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| '''Figure 7. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 30 mg/L Ni²⁺. | | '''Figure 7. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 30 mg/L Ni²⁺. | ||
The graph shows the percentage of Ni²⁺ concentration 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). | The graph shows the percentage of Ni²⁺ concentration 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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| + | | <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> | ||
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| + | | '''Figure 8. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 50 mg/L Ni²⁺. | ||
| + | The graph shows the percentage of Ni²⁺ concentration 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). | ||
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Revision as of 02:29, 2 October 2024
ribozyme+RBS+Hpn+stem-loop
Introduction
This composite part is composed of Hpn coding sequence (CDS), wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting BBa_K4765020 before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.[1] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.[2] In 2023, we extensively tested various stem-loops using BBa_K4765129. For parts we made this year, this strong protective stem-loop sequence was used.
As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used T7 RBS, from bacteriophage T7 gene 10.[3] It is an intermediate strength RBS according to our 2022 results, which allows us to change it to a weaker J6 RBS or a stronger B0 RBS if needed, enabling flexible protein expression levels between various ribozyme connected parts.
This His-rich putative nickel storage protein plays a crucial role in nickel detoxification. Hpn may sequester metals that accumulate internally via a passive equilibrium mechanism (from a high external metals environment).[4]
Usage and Biology
The Hpn can reduce the toxicity of nickel to the E.coli cell.
Get details in BBa_K5115063.
Characterization
Growth curve of E.coli
Before we test the nickel absorption ability of the E.coli, we must make sure that the Ecoli can survive in the waste water. Hpn being the major detoxifying part, we launched experiments on E. coli expressing Hpn compared to E. coli without Hpn expression in mediums containing different concentration of nickel ions.
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| Figure 1: Comparison of E. coli Growth curve with and without Hpn in 20 mg/L Ni²⁺
The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing Hpn compared to E. coli without Hpn expression in a medium containing 20 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without Hpn expression under the same conditions. |
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| Figure 2: Comparison of E. coli Growth curve with and without Hpn in 50 mg/L Ni²⁺
The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing Hpn compared to E. coli without Hpn expression in a medium containing 50 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without Hpn expression under the same conditions. |
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| Figure 3: Comparison of E. coli Growth curve with and without Hpn in 100 mg/L Ni²⁺
The graph illustrates the effect of Ni²⁺ on the growth of E. coli expressing Hpn compared to E. coli without Hpn expression in a medium containing 100 mg/L Ni²⁺ (E.coli strain: BL21 DE3, induced with 1 mM IPTG). The optical density (OD₆₀₀) of the initial bacterial suspension was adjusted to 0.5. E. coli growth was measured by OD₆₀₀, and the bacterial counts were calculated using a standard conversion, where OD₆₀₀ = 1 corresponds to 5.39 × 10⁸ cells. The results indicate that E. coli expressing Hpn has greater tolerance to Ni²⁺, exhibiting higher growth rates than E. coli without Hpn expression under the same conditions. |
Ni absorption experiment
In order to accurately measure the concentration of nickel ions, we adopted the spectrophotometry and used dimethylglyoxime as a color developer, which can specifically bind with Ni. For more details about its protocol, please visit our experiments wiki.
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| Figure 4. Close-up view of the spectrophotometer we used.
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| Figure 5. Every centrifuge tube contained a medium waiting to be made spectrophotometric.
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Ni absorption with a single part introduced
Multiple proteins are used in our design. We launched independent experiments to test their nickel absorbing ability.
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| Figure 6. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 20 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ concentration 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 7. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 30 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ concentration 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 8. Comparison of Ni²⁺ Uptake Efficiency by Different E. coli in 50 mg/L Ni²⁺.
The graph shows the percentage of Ni²⁺ concentration 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). |
Ni absorption with two parts combined
Ni absorption with three parts combined
Ni absorption with four parts combined
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
- ↑ Eiler, D., Wang, J., & Steitz, T. A. (2014). Structural basis for the fast self-cleavage reaction catalyzed by the twister ribozyme. Proceedings of the National Academy of Sciences, 111(36), 13028–13033.
- ↑ Liu, Y., Wu, Z., Wu, D., Gao, N., & Lin, J. (2022). Reconstitution of Multi-Protein Complexes through Ribozyme-Assisted Polycistronic Co-Expression. ACS Synthetic Biology, 12(1), 136–143.
- ↑ The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli. Olins PO, Devine CS, Rangwala SH, Kavka KS. Gene, 1988 Dec 15;73(1):227-35.
- ↑ Maier, R. J., Benoit, S. L., & Seshadri, S. (2007). Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 20(3–4), 655–664.