Difference between revisions of "Part:BBa K5115068"
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===Characterization===
 
===Characterization===
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====Agarose gel electrophoresis=====
 
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We firstly make sure that our genes are successfully introduced into the ''E.coli''
 
  
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In our design, we choose to remove the subunit csoS3 from the cso operon. Previous studies have shown that it is not essential for carboxysome assembly or function. Deleting this subunit can release some burden of the engineered E.coli<ref>Baker, S. H., Williams, D. S., Aldrich, H. C., Gambrell, A. C., & Shively, J. M. (2000). Identification and localization of the carboxysome peptide Csos3 and its corresponding gene in Thiobacillus neapolitanus. Archives of Microbiology, 173(4), 278–283.</ref>.
 
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| <html><img style="width:400px" src="https://static.igem.wiki/teams/5115/registry/nikbedca.png" alt="contributed by Fudan iGEM 2024"></html>
 
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| '''Figure 1. Agarose gel electrophoresis of PCR products amplified from ''E. coli'' (DH5α) colonies.
 
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M: DNA Marker. (A) Lanes 1-8: Amplification of specific regions corresponding to csoS2, csoS3, csoS4A, csoS4B, csoS1C, csoS1A, csoS1B, and csoS1D, demonstrating the presence of the expected subunits derived from the α-carboxysome plasmid. (B) Lanes 1-8: Primers as in (A) were used for amplification. Please note no specific band in lane 2, which is due to the removal of csoS3 from the operon. Also, bands in (B) 3-8 are all smaller than (A) 3-8. Primers for these PCR are listed on https://2024.igem.wiki/fudan/parts.
 
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Revision as of 16:53, 1 October 2024


mineral, nickle module

contributed by Fudan iGEM 2023

Introduction

This part is made up of BBa_K5115038(ribozyme connected: MTA, Hpn, RcnR_C35L) and BBa_K5115086(NixA-F1v) wrapped by ribozyme-assisted polycistronic co-expression system (pRAP) sequences. In BBa_K5115038, the three proteins each are wrapped with the same structure.

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.

With this design, we achieve co-expression of MTA, Hpn, RcnR C35L and NixA-F1vat similar level.

MTA, 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.[4] 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.[5] 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.[6] 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.[7] 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.

Hpn is a 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)[8].

NixA-F1v is F1v linking with NixA's C end. 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[9]. 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.[10]

Usage and Biology

This part is designed to raise the absorption of nickel ions and decrease nickel's toxicity to the E.coli, 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 935
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 198
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. ↑ 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.
  2. ↑ 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.
  3. ↑ 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.
  4. ↑ 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.
  5. ↑ 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.
  6. ↑ 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.
  7. ↑ 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.
  8. ↑ 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.
  9. ↑ 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.
  10. ↑ 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.