Living Materials Constructed with Dynamic Covalent Interface between Synthetic Polymers and B. subtilis

With advances in the field of synthetic biology increasingly allowing us to engineer living cells to perform intricate tasks, incorporating these engineered cells into the design of synthetic polymeric materials will enable programming materials with a wide range of biological functionalities. However, employable strategies for the design of synthetic polymers that form a well-defined interface with living cells and seamlessly integrate their functionalities in materials are still largely limited. Herein, we report the first example of living materials constructed with a dynamic covalent interface between synthetic polymers and living B. subtilis cells. We showedthat 3-acetamidophenylboronic acid (APBA) and polymers of APBA (pAPBA) form dynamic covalent bonds with available diols on the B. subtilis cell surface. Importantly, pAPBA binding to B. subtilis shows a multivalent effect with complete reversibility upon addition of competitive diol species, such as fructose and sorbitol. On the basis of these findings, we constructed telechelic block copolymers with pAPBA chain ends that crosslink B. subtilis cells and produced self- standing living materials. We further demonstrated that the encapsulated cells could be retrieved upon immersing these materials in solutions containing competitive diols and further subjected to biological analyses. This work establishes the groundwork for building a myriad of synthetic polymeric materials integrating engineered living cells and provides a platform for understanding the biology of cells confined within materials.

Analysis of 5′-NAD capping of mRNAs in dormant spores of Bacillus subtilis

ABSTRACT Spores of Gram-positive bacteria contain 10s–1000s of different mRNAs. However, Bacillus subtilis spores contain only ∼ 50 mRNAs at > 1 molecule/spore, almost all transcribed only in the developing spore and encoding spore proteins. However, some spore mRNAs could be stabilized to ensure they are intact in dormant spores, perhaps to direct synthesis of proteins essential for spores’ conversion to a growing cell in germinated spore outgrowth. Recent work shows that some growing B. subtilis cell mRNAs contain a 5′-NAD cap. Since this cap may stabilize mRNA in vivo, its presence on spore mRNAs would suggest that maintaining some intact spore mRNAs is important, perhaps because they have a translational role in outgrowth. However, significant levels of only a few abundant spore mRNAs had a 5′-NAD cap, and these were not the most stable spore mRNAs and had likely been fragmented. Even higher levels of 5′-NAD-capping were found on a few low abundance spore mRNAs, but these mRNAs were present in only small percentages of spores, and had again been fragmented. The new data are thus consistent with spore mRNAs serving only as a reservoir of ribonucleotides in outgrowth.