A living and functional semi- interpenetrating polymer network material fabricated by engineered bacteria

In this paper, we reported the fabrication of functional and biocompatible sIPN by integrating the non-living polymeric microcapsules and engineered bacteria. Especially, we adopt the platform to protect gut microbiota in mice from antibiotic-mediated perturbations.

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An interpenetrating polymer network (IPN) is a combination of two or more polymer networks that are interlaced with each other. If one of the polymers is not fully crosslinked, a semi-IPN (sIPN) results. The beauty of the system is that it can integrate the advantages from both components. For example, a semi-IPN composed of a temperature-sensitive component and a pH-sensitive component can respond to both cues. Besides, the interlacing between two or more components can reinforce or modulate the physical properties, such as mechanical strength of resultant material. Since their invention, most sIPN is assembled by chemical methods, making it difficult to incorporate biocompatible functions. Advances in protein engineering have now made it possible to engineer protein tags that can react and form covalent bonds under mild conditions. For example, protein polymer or hydrogels can be created by using proteins with reactive tags. However, this line of research is almost entirely done by using purified protein components and the fabrication process often entails multiple expensive and time-consuming steps. Importantly, the sIPN fabricated by chemical processes or protein material prepared by the purified components cannot self-repair: once lost (e.g. due to external perturbations), their functions cannot recover.

The past 20 years has witnessed tremendous progress in synthetic biology, particularly in the ability to assemble complex gene circuits and to program certain well-defined dynamic functions. A few recent examples have demonstrated the use of engineered bacteria to fabricate materials. However, most of these efforts focused on a proof-of-concept demonstration of the fabrication process. Also, the resulting materials do not exploit unique features of the living cells, though the cells are critical for fabrication.

In this work, we demonstrate the engineering of living, functional sIPN consisting of engineered bacteria and non-living components. In particular, the fabrication process is driven by engineered bacteria encapsulated in a polymeric microcapsule, which serves as the initial scaffold. The bacteria grow and undergo programmed lysis in a density-dependent manner, releasing protein monomers decorated with functional tags. Those protein monomers polymerize with each other to form a layer of protein mesh that is interlaced with the polymeric scaffold (Figure 1). The formation of sIPN serves the dual purposes of enhancing the mechanical property of the living materials and anchoring effector proteins for diverse applications. Due to constitutive assembly of the protein components (thanks to the living cells), the material is resilient in its ability to recover its programmed function after transient or constant perturbations: i.e. it can self-repair during and post perturbations.

Figure 1. Living fabrication of sIPN by engineered bacteria. Credits to Xiao Peng in Rice University. 

By design, the process is biocompatible and versatile to incorporate functional proteins into the sIPN by simply encoding the desired protein sequence into the downstream of the monomer region. To this end, we incorporate an enzyme (Beta-lactamase, Bla) that can degrade antibiotics (Beta-lactams) into the sIPN and demonstrate the engineering of living sIPN to efficiently protect gut microbiota from an antibiotic-mediated perturbation. Using pure Bla is an option but free Bla is prone to breakdown (e.g., by proteinases) inside the gut, reducing the efficiency of protection. Our living material can increase treatment efficacy presumably because of the continual production of Bla by the living bacteria as well enhanced Bla stability due to formation of sIPN.

The system allows versatile functionalization of the scaffold polymer by incorporating different effector proteins, depending on the application context. We foresee that the potential implications range from biomanufacturing by incorporating multiple enzymes for cascade catalysis, environmental clean-up or recycling by incorporating toxin degrading enzymes or metal-binding proteins, to disease diagnosis and treatment by incorporating protein therapeutics.

 

Curious for more? You can read our full paper “Living fabrication of functional semi-interpenetrating polymeric materials ” published in Nature Communications.

Zhuojun Dai

Associate Professor, Shenzhen Institute of Advanced Technology, CAS