Hybrid Living Capsules Autonomously Produced by Engineered Bacteria.

Daniel P Birnbaum,Blaise L Tardy,Avinash Manjula-Basavanna,Neel S Joshi,Anton Kan

doi:10.1002/advs.202004699

Abstract

Bacterial cellulose (BC) has excellent material properties and can be produced sustainably through simple bacterial culture, but BC‐producing bacteria lack the extensive genetic toolkits of model organisms such as Escherichia coli (E. coli). Here, a simple approach is reported for producing highly programmable BC materials through incorporation of engineered E. coli. The acetic acid bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in droplets of glucose‐rich media to produce robust cellulose capsules, which are then colonized by the E. coli upon transfer to selective lysogeny broth media. It is shown that the encapsulated E. coli can produce engineered protein nanofibers within the cellulose matrix, yielding hybrid capsules capable of sequestering specific biomolecules from the environment and enzymatic catalysis. Furthermore, capsules are produced which can alter their own bulk physical properties through enzyme‐induced biomineralization. This novel system uses a simple fabrication process, based on the autonomous activity of two bacteria, to significantly expand the functionality of BC‐based living materials.

Highlights

This material is protected by copyright and other intellectual property rights, and duplication or sale of all or part of any of the repository collections is not permitted, except that material may be duplicated by you for your research use or educational purposes in electronic or print form
Bacterium Gluconacetobacter hansenii is cocultured with engineered E. coli in There is great potential for the developdroplets of glucose-rich media to produce robust cellulose capsules, which are colonized by the E. coli upon transfer to selective lysogeny broth media
We chose to produce the Bacterial cellulose (BC) material as a hollow spherical capsule rather than the more conventional pellicle format in the hopes of creating a material with topologically distinct regions, with G. hansenii primarily occupying the “outer” shell composed of a dense cellulose matrix, and E. coli occupying the “inner” core

Summary

Results

We chose to produce the BC material as a hollow spherical capsule rather than the more conventional pellicle format in the hopes of creating a material with topologically distinct regions, with G. hansenii primarily occupying the “outer” shell composed of a dense cellulose matrix, and E. coli occupying the “inner” core. Following the incubation in HS, the newly formed capsules were transferred to lysogeny broth (LB) media (containing antibiotic to select for engineered E. coli) and incubated under shaking conditions to proliferate E. coli inside the capsules (Figure 1A). When incubated in LB with the addition of 0.1% w/w arabinose, the capsules exhibited a bright red appearance due to the production of RFP in the engineered E. coli (Figure 2A) This demonstrated that the plasmid was functional in the encapsulated E. coli, while allowing us to clearly visualize the growth of E. coli colonies inside the capsules. To test the efficiency of E. coli encapsulation within the cellulose matrix following the growth period in LB, we agitated capsules containing BL21/pBbA8k-RFP cells in PBS for 24 h and found that ≈95% of the E. coli cells were retained in the capsule, while the remaining 5% leaked into the PBS supernatant (Figure S1, Supporting Information)

Coculture Dynamics during Capsule Production

Functional Hybrid Capsules Containing Engineered Curli Nanofibers

Programmable Biomineralization of Capsules

Conclusion

Experimental Section

Data Availability Statement