Layer-by-layer assembly of bioengineered flagella protein nanotubes

Flagella nanotubes present on the surface of E. coli bacteria were bioengineered to display arginine-lysine and glutamic acid-aspartic acid peptide loops. These protein bionanotubes were demonstrated to self-assemble, layer-by-layer, by atomic force microscopy (AFM) on gold-coated mica and quartz surfaces. Flagella with arginine-lysine loops were assembled in a bottom-up manner on a gold-coated mica surface by employing the molecular complementarity of the biotin-streptavidin interaction. Self-assembled monolayers of alkylamines on the gold surface were derivatized with biotin, followed by binding of streptavidin to the biotinylated surface. The amine groups of the flagella peptide loops were chemically attached to biotin through a polyethyleneoxide spacer and paired with streptavidin on the gold surface. This process could be repeated to generate multiple layers of flagella. Flagella with glutamic acid-aspartic acid peptide loops were self-assembled on quartz surfaces by electrostatic attraction to protonated amine groups. The quartz surface was silanized to obtain amine groups, which were used to assemble the first layer of glutamic acid-aspartic acid peptide loop flagella nanotubes. This layer was covered with polyethyleneimine through electrostatic attraction and employed to assemble a second layer of flagella. The self-assembled glutamic acid-aspartic acid flagella were also used to demonstrate the biomineralization of CaCO 3. The layer-by-layer self-assembly employing electrostatic attraction yielded a more uniform layer of flagella than the one obtained with the molecular complementarity of the biotin-streptavidin pair.