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. 2021 Dec 8;12:7133. doi: 10.1038/s41467-021-27467-2

Fig. 8. ELM biocomposite regeneration and proof-of-concept for material functionalization.

Fig. 8

a Silica biocomposite material regeneration was tested by re-inoculating cultures with a piece of a silica plug fabricated from B. subtilis ΔlytC ΔflhG cultures co-expressing EutM-CotB and EutM-SpyCatcher and HagT209CSpyTag588, biomineralized with 200 mM silica (see Fig. 6) and cured for 24 h at 25 °C. Cultures were regrown (SMM, 20 °C, 48 h) with (+ induced) and without (- uninduced) cumate induction of protein expression. b Expression and secretion of EutM scaffold building blocks by the induced, regenerated cultures was confirmed by SDS-PAGE analysis of culture supernatant (A) and urea solubilized scaffolds (B). All fractions were concentrated 10-fold by TCA precipitation prior to SDS-PAGE analysis. c Silica biomineralization into a cross-linked biocomposite material was confirmed for the induced, regenerated cultures after 1 h at 20 °C with 100 mM silica in 6-well plates as in Fig. 6. d Proof-of-concept incorporation of additional functions into the silica ELM was demonstrated by the co-cultivation of two strains, one strain co-expresses scaffold building blocks and SpyTagged flagellin, while a second strain co-expresses a purple chromophore protein and SpyTagged flagellin. In the control co-culture, the plasmid for scaffold building block secretion in one strain was replaces with an empty plasmid. Strains were inoculated at a 1:1 ratio and cultures grown under inducing conditions for biomineralization as described above. Only co-cultures that co-expressed EutM-CotB and EutM-SpyCatcher formed an aggregated silica material with embedded purple cells. e A block of a purple, silica biocomposite (15 mm × 30 mm) was then fabricated from co-cultures of engineered strains co-expressing scaffold building blocks and the purple protein together with SpyTagged flagellin. A solid material was formed with 200 mM silica after curing for 24 h at 25 °C. All results shown are representative of three independent biological samples. Source data are provided as a Source Data file.