(67b) Dynamic CRISPR-Based Genetic Programs in Microbe-Laden Hydrogels for Bioproduction

There has been a growing interest in using engineered living materials (ELMs) for on-demand bioproduction as compartmentalized bacteria are reported to sustain long-term metabolic activity¹. However, the extent to which genetic activity can be sustained in compartmentalized bacteria has not been fully explored. In this work, we applied CRISPR-based expression tools on E. coli MG1655 compartmentalized in Pluronic F127-bisurethane methacrylate (F127-BUM) hydrogels to investigate the ability of hydrogel-immobilized microbes in sustaining expression of genetic programs in continuous culture conditions. By directly measuring gene expression dynamics in hydrogels, we found that inducible gene expression is sustained for multiple weeks. This study also reveals that even after 19 days of continuous culture, compartmentalized bacteria retain >50% of their original inducible gene expression capacity. The hydrogel-compartmentalized bacteria also maintain responsiveness to multiple induction cycles, retaining >46% of the original gene expression capacity following a second induction cycle. Using the knowledge of dynamic genetic response in microbe-laden hydrogels, we successfully predicted bioproduction phenotypes arising from inducible CRISPR transcriptional activation (CRISPRa) programs controlling a multi-gene biosynthetic pathway of pteridine-derivatives. This work demonstrates that hydrogel-immobilized microbes can retain expression of CRISPR-based genetic programs for sustained bioproduction, foreshadowing the creation of new generation ELMs for controllable, scalable, and versatile on-demand bioproduction.

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