Difference between revisions of "Part:BBa K1151001"

Revision as of 11:28, 24 September 2024

Improved by Fudan iGEM 2024

To form a programmable self-assembling biofilm, different strengths of Ag/Nb pairs are required. In our project, we employed the surface display system neae(intimin) to present three pairs of Ag/Nb on the E. coli 's cell membrane and assessed their strengths. Simultaneously, we connected lectins LCA and MVN to the C-terminus of intimin, facilitating the binding between E. coli and cyanobacteria. As a result, we established a stable symbiotic platform between cyanobacteria and E. coli.

Improved part

Our improved part is BBa_K4765106 (Twister P1 + T7_RBS + intimin-Nb3 fusion + stem-loop),BBa_K4765109 (Twister P1 + T7_RBS + intimin-MVN fusion + stem-loop) and BBa_K4765110 (Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop). We not only introduce three independent antigen-nanobody pairs and two lectins into the synthetic adhesion system but as well construct these parts into our ribozyme-assisted polycistronic co-expression system.

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