Difference between revisions of "Part:BBa K5115068"
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===Introduction=== | ===Introduction=== | ||
| + | This part is made up of MTA, Hpn, RcnR_C35L and NixA-F1v, all of the proteins constructed into our ribozyme-assisted polycistronic co-expression system. | ||
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| + | MTA [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050], sourced from ''Pisum sativum'', is a cysteine-rich protein known for its high binding affinity for various heavy metals, including nickel. By sequestering excess nickel ions, MTA further reduces the potential cytotoxic effects associated with elevated nickel levels.<ref>Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences: CMLS, 59(4), 627–647.</ref> Hpn [https://parts.igem.org/Part:BBa_K1151001 BBa_K1151001], derived from Helicobacter pylori, is characterized by its high histidine content. Its structure allows it to exist in various multimeric forms in solution. The primary function of Hpn is to bind nickel ions, with the ability to sequester up to five Ni²⁺ ions per monomer in a pH-dependent manner (optimal at pH 7.4).By binding and storing excess nickel, Hpn prevents harmful interactions between nickel ions and cellular machinery, thereby promoting the survival and functionality of E. coli in nickel-rich conditions.<ref>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.</ref> This dual strategy, combining Hpn and MTA, enhances the overall nickel absorptivity of our engineered bacteria while simultaneously minimizing the harmful effects of nickel accumulation. | ||
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| + | RcnR_C35L [https://parts.igem.org/Part:BBa_K5115000 BBa_K5115000],a nickel-responsive transcriptional regulator, can optimize nickel uptake by modulating gene expression. RcnR is a tetrameric transcriptional repressor that responds to the binding of Ni(II) ions by releasing DNA, resulting in the expression of RcnA, which is responsible for nickel export from the cell.<ref>Huang, H.-T., & Maroney, M. J. (2019). Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage. Inorganic Chemistry, 58(20), 13639–13653.</ref> By inhibiting RcnA, RcnR ensures that intracellular nickel ion concentrations remain elevated, thereby enhancing the effectiveness of our nickel absorption system. As to the nickel transporting protein, we choose to use NixA [https://parts.igem.org/Part:BBa_K5115071 BBa_K5115071], a monomeric protein which is specifically adapted for nickel transport.<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> In our design, we don't simply introduce NixA into the ''E.coli''. Instead, we linked it with F1v [https://parts.igem.org/Part:BBa_K5115085 BBa_K5115085], optimizing NixA's dimerization to enhance its transporting ability. | ||
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| + | Based on the ribozyme-assisted polycistronic co-expression system, all of the proteins above can express at a equalized level. To learn more about our pRAP system, please check [https://2022.igem.wiki/fudan/parts part wiki of 2022 Fudan iGEM]. | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This part is designed to raise the absorption of nickel ions and decrease nickel's toxicity, which is the basic working module of our project. This part ensures that the nickel ions in the wastewater can be locked inside the ''E.coli'', laying the foundation for the following processing steps. | ||
===Characterization=== | ===Characterization=== | ||
Revision as of 03:28, 30 September 2024
mineral, nickle module
Introduction
This part is made up of MTA, Hpn, RcnR_C35L and NixA-F1v, all of the proteins constructed into our ribozyme-assisted polycistronic co-expression system.
MTA BBa_K5115050, sourced from Pisum sativum, is a cysteine-rich protein known for its high binding affinity for various heavy metals, including nickel. By sequestering excess nickel ions, MTA further reduces the potential cytotoxic effects associated with elevated nickel levels.[1] Hpn BBa_K1151001, derived from Helicobacter pylori, is characterized by its high histidine content. Its structure allows it to exist in various multimeric forms in solution. The primary function of Hpn is to bind nickel ions, with the ability to sequester up to five Ni²⁺ ions per monomer in a pH-dependent manner (optimal at pH 7.4).By binding and storing excess nickel, Hpn prevents harmful interactions between nickel ions and cellular machinery, thereby promoting the survival and functionality of E. coli in nickel-rich conditions.[2] This dual strategy, combining Hpn and MTA, enhances the overall nickel absorptivity of our engineered bacteria while simultaneously minimizing the harmful effects of nickel accumulation.
RcnR_C35L BBa_K5115000,a nickel-responsive transcriptional regulator, can optimize nickel uptake by modulating gene expression. RcnR is a tetrameric transcriptional repressor that responds to the binding of Ni(II) ions by releasing DNA, resulting in the expression of RcnA, which is responsible for nickel export from the cell.[3] By inhibiting RcnA, RcnR ensures that intracellular nickel ion concentrations remain elevated, thereby enhancing the effectiveness of our nickel absorption system. As to the nickel transporting protein, we choose to use NixA BBa_K5115071, a monomeric protein which is specifically adapted for nickel transport.[4] In our design, we don't simply introduce NixA into the E.coli. Instead, we linked it with F1v BBa_K5115085, optimizing NixA's dimerization to enhance its transporting ability.
Based on the ribozyme-assisted polycistronic co-expression system, all of the proteins above can express at a equalized level. To learn more about our pRAP system, please check part wiki of 2022 Fudan iGEM.
Usage and Biology
This part is designed to raise the absorption of nickel ions and decrease nickel's toxicity, which is the basic working module of our project. This part ensures that the nickel ions in the wastewater can be locked inside the E.coli, laying the foundation for the following processing steps.
Characterization
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 935
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 198
- 1000COMPATIBLE WITH RFC[1000]
References
- ↑ Coyle, P., Philcox, J. C., Carey, L. C., & Rofe, A. M. (2002). Metallothionein: The multipurpose protein. Cellular and Molecular Life Sciences: CMLS, 59(4), 627–647.
- ↑ 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.
- ↑ Huang, H.-T., & Maroney, M. J. (2019). Ni(II) Sensing by RcnR Does Not Require an FrmR-Like Intersubunit Linkage. Inorganic Chemistry, 58(20), 13639–13653.
- ↑ 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.