Difference between revisions of "Part:BBa K1151001"
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===Improved by Fudan iGEM 2024 === | ===Improved by Fudan iGEM 2024 === | ||
This part is called hpn for short. The 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).<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> The hpn working together with [https://parts.igem.org/Part:BBa_K5115000 BBa_K5115000(RcnR_C35L)]and [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050(MTA)], they can raise the absorptivity of nickel ion as well as reduce the harm nickel brings to our engineering bacteria. | This part is called hpn for short. The 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).<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> The hpn working together with [https://parts.igem.org/Part:BBa_K5115000 BBa_K5115000(RcnR_C35L)]and [https://parts.igem.org/Part:BBa_K5115050 BBa_K5115050(MTA)], they can raise the absorptivity of nickel ion as well as reduce the harm nickel brings to our engineering bacteria. | ||
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| + | When inserted into a polycistronic sequence, the expression of hpn could be influenced by the self-interaction of the polycistron. Instead of assembling CDSs sequentially, we construct a ribozyme-assisted polycistronic co-expression system (pRAP) to the hpn. By inserting [https://parts.igem.org/Part:BBa_K4765020 ribozyme sequences] before hpn, the RNA sequences of Twister ribozyme conduct self-cleaving in the mRNA, avoiding the self-interaction of the polycistron.<ref>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.</ref> To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of hpn.<ref>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.</ref> | ||
====Improved part==== | ====Improved part==== | ||
| − | Our improved part is [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop)]. We | + | Our improved part is [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop)]. We introduced this ribozyme-assisted polycistronic co-expression system in [https://2022.igem.wiki/fudan/parts 2022] to hpn, ensuring a satisfying expression of this protein. The hpn has been integrated into many parts in [2024.igem.org/fudan/parts 2024 Fudan iGEM parts], [https://parts.igem.org/Part:BBa_K5115068 BBa_K5115068(mineral, nickel module)] being the most important one. |
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Revision as of 01:34, 1 October 2024
Improved by Fudan iGEM 2024
This part is called hpn for short. The 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).[1] The hpn working together with BBa_K5115000(RcnR_C35L)and BBa_K5115050(MTA), they can raise the absorptivity of nickel ion as well as reduce the harm nickel brings to our engineering bacteria.
When inserted into a polycistronic sequence, the expression of hpn could be influenced by the self-interaction of the polycistron. Instead of assembling CDSs sequentially, we construct a ribozyme-assisted polycistronic co-expression system (pRAP) to the hpn. By inserting ribozyme sequences before hpn, the RNA sequences of Twister ribozyme conduct self-cleaving in the mRNA, avoiding the self-interaction of the polycistron.[2] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of hpn.[3]
Improved part
Our improved part is BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop). We introduced this ribozyme-assisted polycistronic co-expression system in 2022 to hpn, ensuring a satisfying expression of this protein. The hpn has been integrated into many parts in [2024.igem.org/fudan/parts 2024 Fudan iGEM parts], BBa_K5115068(mineral, nickel module) being the most important one.
Histidine-rich metal-binding protein
So called for emphasize its origins in Helicobacter pylori and its avidity for nickel. Consisting of 60 aminoacids, the protein is rich in histidine (28 residues, 46.7 %) and contains short repeating motifs, it exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Hpn can bind tightly and reversibly up to five Ni2+ ions per each monomer of 7 kDa in a pH-dependent manner (pH 7.4 ). In H. pylori play an important role in storing nickel required to the survival of the bacterium.
Boiling prep and digestion with restriction enzymes
Once inserted the gene coding for Hpn in a PSB1C3 plasmid (digestion and ligation), we transformed DH5a cells with the construct and then we recovered the ligated plasmid by boiling prep. We then proceeded with the digestion with EcoRI and PstI to check.
Figure 1: Digestion result of boiling prep plasmids.
PCR with VF2 and VR2 primers
Figure 2: PCR results.
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]
- ↑ 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.
- ↑ 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.