Two New Plasmid Post-segregational Killing Mechanisms for the Implementation of Synthetic Gene Networks in Escherichia coli
Alex J.H. Fedorec,
Tanel Ozdemir,
Anjali Doshi,
Yan-Kay Ho,
Luca Rosa,
Jack Rutter,
Oscar Velazquez,
Vitor B. Pinheiro,
Tal Danino,
Chris P. Barnes
Affiliations
Alex J.H. Fedorec
Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK; Centre for Mathematics, Physics and Engineering in the Life Sciences and Experimental Biology, University College London, London WC1E 6BT, UK; Corresponding author
Tanel Ozdemir
Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK
Anjali Doshi
Department of Biomedical Engineering, Columbia University, New York City, NY 10027, USA
Yan-Kay Ho
Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK
Luca Rosa
Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK
Jack Rutter
Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK
Oscar Velazquez
Department of Biomedical Engineering, Columbia University, New York City, NY 10027, USA
Vitor B. Pinheiro
Institute of Structural and Molecular Biology, University College London, London WC1E 6BT, UK; KU Leuven Rega Institute for Medical Research, Herestraat, 49 Box 1030, 3000 Leuven, Belgium
Tal Danino
Department of Biomedical Engineering, Columbia University, New York City, NY 10027, USA; Data Science Institute, Columbia University, New York, NY 10027, USA; Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA
Chris P. Barnes
Department of Cell and Developmental Biology, University College London, London WC1E 6BT, UK; Department of Genetics, Evolution and Environment, University College London, London WC1E 6BT, UK; Corresponding author
Summary: Plasmids are the workhorse of both industrial biotechnology and synthetic biology, but ensuring they remain in bacterial cells is a challenge. Antibiotic selection cannot be used to stabilize plasmids in most real-world applications, and inserting dynamical gene networks into the genome remains challenging. Plasmids have evolved several mechanisms for stability, one of which, post-segregational killing (PSK), ensures that plasmid-free cells do not survive. Here we demonstrate the plasmid-stabilizing capabilities of the axe/txe toxin-antitoxin system and the microcin-V bacteriocin system in the probiotic bacteria Escherichia coli Nissle 1917 and show that they can outperform the commonly used hok/sok. Using plasmid stability assays, automated flow cytometry analysis, mathematical models, and Bayesian statistics we quantified plasmid stability in vitro. Furthermore, we used an in vivo mouse cancer model to demonstrate plasmid stability in a real-world therapeutic setting. These new PSK systems, plus the developed Bayesian methodology, will have wide applicability in clinical and industrial biotechnology. : Biological Sciences; Gene Network; Bioengineering Subject Areas: Biological Sciences, Gene Network, Bioengineering
