Difference between revisions of "Part:BBa K1151001"

Revision as of 15:13, 26 September 2024

Improved by Fudan iGEM 2024

This part is called hpn for short. The His-rich putative nickel storage proteins Hpn plays a 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.

Improved part

Our improved part is BBa_K5115036 (Ribozyme + RBS + Hpn + stem-loop). We construct this part into our ribozyme-assisted polycistronic co-expression system, which ensures a satisfying expression of hpn.

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

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.