Figure 2: Genetic engineering of the BIND platform.
From: Programmable biofilm-based materials from engineered curli nanofibres
(a) A library of CsgA fusion mutants in which the MBD peptide insert (purple) was placed at the N- or C-terminus of the curlin repeat domains (red) and flanked by a six-residue linker, two-residue linker or no linker (green). (b) The MBD insert library was transformed into LSR10 (MC4100, ΔcsgA) cells and streaked onto YESCA-Congo Red induction plates. Red staining indicates amyloid production. (c) A representative set of culture spots of a BIND library consisting of 12 various functional peptides on YESCA-Congo Red induction plates (enumerated in Table 1). (d) Quantitative Congo Red values were obtained from quadruplicate YESCA-CR spotted cultures using intensity quantification (ImageJ) measurements of the relative amyloid produced for each CsgA-peptide fusion, normalized to wild-type CsgA. Standard error is shown. (e) Whole-cell filtration ELISA using an anti-CsgA antibody (black bars); secondary antibody–only controls are shown as grey bars. Each experiment was performed in triplicate and standard error is shown. FE-SEM images of the peptide fusion BIND library transformed into LSR10 (MC4100, ΔcsgA) cells with no CsgA (f), wild-type CsgA (g) and the BIND peptide panel (see Table 1): HIS (h), GBP (i), FLAG (j), CNBP (k), A3 (l), CLP12 (m), QBP1 (n), SpyTag (o), MBD (p), CT43 (q), AFP8 (r) and Mms6 (s). All scale bars are 1 μm.
