Developing a new cellular platform can enable new biotechnological applications powered by the unique characteristics of a given cell chassis. The field of synthetic biology has notable examples of the development of transformational cellular platforms such as synthetic bacteria (1), artificial cells (2), and reprogrammed mammalian cells (3). These cellular systems fall in either the category of living cells, which are complex and difficult to control, or biomimetic entities, which are simple and have predictable functions. Our interest in this field started with the publication of a study demonstrating the use of intracellular hydrogelation in mammalian cells to enhance the structural integrity of the cell and provide mechanical support for plasma membranes (4). We hypothesized that with careful experimentation and precise bioengineering, there might be a way to use intracellular hydrogelation to tackle two of the main hurdles with commonly used cellular platforms: uncontrolled proliferation, and susceptibility to extracellular environmental stressors.
As an emerging cellular engineering method, introducing synthetic polymer matrices into the cellular cytosol enables the modification and control of cellular states, facilitating the development of semi-living biomaterials (5). In our 2023 paper (6), we introduced Cyborg Bacterial Cells as a new cellular platform developed at the intersection of synthetic biology and materials science. These semi-living entities do not divide, are resistant to extracellular stressors, and can be reprogrammed using genetic constructs to give them precise functionalities. Their potential applications include biosensing, drug delivery, and their use as therapeutic agents against cancer. In our recently published protocol (7), we aim to make the development of Cyborg Bacterial Cells more accessible to the scientific community by introducing a detailed step-by-step guide to produce these semi-living micromachines together with precise validation metrics to benchmark and test the efficiency of the Cyborg Bacterial Cell preparation.
The idea of creating Cyborg Bacterial Cells using intracellular hydrogelation was born out of a collaboration between the laboratories of Cheemeng Tan at the University of California, Davis, and Che-Ming Jack Hu from Academia Sinica in Taiwan. After a few key experiments testing the feasibility of intracellular bacterial hydrogelation, the group embarked on trying to find a “goldilocks zone” where bacteria could be hydrogelated, unable to divide and show metabolic activity. Luis E. Contreras-Llano and Yu-Han Liu were the researchers in both laboratories who carried out the foundational experiments and demonstrated the reproducibility of the method by independently reproducing the creation of Cyborg Bacterial Cells in two different laboratories at UC Davis (California, USA) and Academia Sinica (Taipei, Taiwan).
After the initial development of the Cyborg Bacterial Cell platform, another pair of researchers at UC Davis, Ofelya Baghdasaryan and Shahid Khan further developed the semi-synthetic cellular chassis, starting with robust validation metrics and methodologies to make the creation of Cyborg Bacterial Cells accessible to other researchers. Eventually, Ofelya, Luis, and Shahid (Fig. 1) teamed up to develop a comprehensive protocol detailing the fabrication of these Cyborg Bacteria that we hope the scientific community can further explore, program, and use. The protocol details the nuances in generating Cyborg Bacterial Cells, using E. coli Nissle 1917 as our example organism. Additionally, we propose six non-exclusive validation options to benchmark Cyborg Bacterial Cell preparations and to guide further development when attempting to extrapolate the protocol to different microorganisms.
Figure 1. Shahid Khan, Ofelya Baghdasaryan, and Luis Eduardo Contreras Llano, the team behind the protocol to create Cyborg Bacterial Cells.
We are incredibly excited about the potential of Cyborg Bacterial Cells. Our original manuscript demonstrated their functionality as biosensors and showed their ability to penetrate cancer cells. Our efforts now focus on developing applications using these semi-living cells as therapeutics and drug-delivery devices. Furthermore, we are investigating the quasi vita state of the Cyborg Cells. Understanding how hydrogelation allows metabolic activity while impeding cell replication could enable a new way to study bacterial morphology and biophysics, contribute to the elucidation of the spatial-temporal organization of the cellular cytoplasm, and deepen our understanding of what life itself means.
References:
Charbonneau, M. R., Isabella, V. M., Li, N. & Kurtz, C. B. Developing a new class of engineered live bacterial therapeutics to treat human diseases. Nat. Commun. 11, 1–11 (2020).
Ding, Y., Contreras-Llano, L. E., Morris, E., Mao, M. & Tan, C. Minimizing Context Dependency of Gene Networks Using Artificial Cells. ACS Appl. Mater. Interfaces 10, 30137–30146 (2018).
Bashor, C. J., Hilton, I. B., Bandukwala, H., Smith, D. M. & Veiseh, O. Engineering the next generation of cell-based therapeutics. Nat. Rev. Drug Discov. 21, 655–675 (2022).
Lin, J. C. et al. Intracellular hydrogelation preserves fluid and functional cell membrane interfaces for biological interactions. Nat. Commun. 10, 1–11 (2019).
Baghdasaryan, O. et al. Synthetic control of living cells by intracellular polymerization. Trends Biotechnol. S0167779923002408 (2023).
Contreras-Llano, L. E. et al. Engineering Cyborg Bacteria Through Intracellular Hydrogelation. Adv. Sci. 2204175, 1–11 (2023).
Baghdasaryan, O., Contreras-Llano, L. E., Khan, S., et al. Fabrication of cyborg bacterial cells as living cell-material hybrids using intracellular hydrogelation. Nat. Prot. (2024).