In vivo modified organic matrix for testing biomineralization-related protein functions in differentiated...

This work reports an in vivo approach for identifying the function of biomineralization-related proteins. Synthetic sequences of n16N, OC-17 and perlucin with signal peptides are produced in a novel Gateway expression system for Dictyostelium under the control of the [ecmB] promoter. A fast and easy scanning electron microscopic screening method was used to differentiate on the colony level between interplay effects of the proteins expressed in the extracellular matrix (ECM). Transformed Dictyostelium, which migrated as multicellular colonies on calcite crystals and left their ECM remnants on the surface were investigated also by energy-dispersive X-ray spectroscopy (EDX). Calcium minerals with and without phosphorous accumulated very frequently within the matrix of the Dictyostelium colonies when grown on calcite. Magnesium containing phosphorous granules were observed when colonies were exposed on silica. The absence of calcium EDX signals in these cases suggests that the external calcite crystals but not living cells represent the major source of calcium in the ECM. Several features of the system provide first evidence that each protein influences the properties of the matrix in a characteristic mode. Colonies transformed with perlucin produced a matrix with cracks on the length scale of a few microns throughout the matrix patch. For colonies with OC-17, almost no cracks were observed, regardless of the length scale. The non-transformed Dictyostelium (Ax3-Orf+) produced larger cracks. The strategy presented here develops the first step toward an efficient eukaryotic screening system for the combinatorial functionalization of materials by bioengineering in close analogy to natural biomineralization concepts.