Difference between revisions of "Part:BBa K1151001"

Revision as of 04:00, 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), together they can trick bacteria to aggressively absorb nickel ion from the environment, as well as reducing the harm nickel brings to the bacteria.

Improved part

Our improved part is BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop). We wrapped hpn coding sequence with 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.^[2] To protect the mono-cistron mRNA from degradation, a stem-loop structure is placed at the 3' end of CDS.^[3] In 2023, we extensively tested various stem-loops using BBa_K4765129.

As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used T7 RBS, from bacteriophage T7 gene 10.^[4] 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.

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE 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.
↑ 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.

[1] 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.

[2] 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.

[3] 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.

[4] 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.

[1]

[2]

[3]

[4]

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 <html><img style="float:left;width:64px;margin-right:2em" src="https://static.igem.wiki/teams/5115/czh/mineral-logo.svg" alt="contributed by Fudan iGEM 2024"></html>
-===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)], together they can trick bacteria to aggressively absorb nickel ion from the environment, as well as reducing the harm nickel brings to the 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 [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====
-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.
+Our improved part is [https://parts.igem.org/Part:BBa_K5115036 BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop)]. We wrapped hpn coding sequence with ribozyme-assisted polycistronic co-expression system (pRAP) sequences. By inserting [https://parts.igem.org/Part:BBa_K4765020 BBa_K4765020] before CDS, the RNA of Twister ribozyme conduct self-cleaving in the mRNA.<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 CDS.<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> In 2023, we extensively tested various [https://2023.igem.wiki/fudan/part-collection/#ribozyme-assisted-polycistronic-co-expression stem-loops] using [https://parts.igem.org/Part:BBa_K4765129 BBa_K4765129].
+As for the ribosome binding sequence (RBS) after the ribozyme and before the CDS, we used [https://parts.igem.org/Part:BBa_K4162006 T7 RBS], from bacteriophage T7 gene 10.<ref>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.</ref> It is an intermediate strength RBS according to [https://2022.igem.wiki/fudan/measurement#optimization our 2022 results], which allows us to change it to a weaker [https://parts.igem.org/Part:BBa_J61100 J6 RBS] or a stronger [https://parts.igem.org/Part:BBa_B0030 B0 RBS] if needed.
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