A modular toolkit for theophylline-dependent synthetic auxotrophs via riboswitch-gated essential genes
- 1Synvivia, Inc., Emeryville, California, United States of America
Abstract
Synthetic auxotrophs are a useful means for developing genetically encoded biocontainment systems. Current methods for developing synthetic auxotrophs are complicated or expensive, limiting access and adoption of biocontainment technologies. To address this gap, we developed a simple and modular platform for creating synthetic auxotrophs based on ligand-dependent translational gating of essential genes. By inserting a theophylline-responsive riboswitch upstream of essential genes in Escherichia coli, we created strains whose viability depends on the presence of theophylline. We systematically applied this approach to 29 essential genes, obtained ligand-dependent growth phenotypes for 19 targets, and found that 18 of these essential genes yielded stringent live-die synthetic auxotrophs. These strains exhibited robust theophylline dependence with escape frequencies ranging from 1 × 10−5 to 1 × 10−6 (most below limit of detection). Our modular design allows for the rapid (<1 week), low-cost, and reliable generation of synthetic auxotrophs. This work introduces ligand-dependent translational control as a new mechanism for engineering synthetic auxotrophy and provides an accessible platform that expands the biocontainment toolkit.
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BibTeX
@article{gonzalezlopez2026modular,
title = {{A modular toolkit for theophylline-dependent synthetic auxotrophs via riboswitch-gated essential genes}},
author = {Carlos Gonzalez-Lopez and Adrian Overly and Saahil Singh and Charlie Huang and Gabriel Lopez},
journal = {PLOS One},
year = {2026},
month = oct,
doi = {10.1371/journal.pone.0360098},
url = {https://doi.org/10.1371/journal.pone.0360098},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A modular toolkit for theophylline-dependent synthetic auxotrophs via riboswitch-gated essential genes
AU - Carlos Gonzalez-Lopez
AU - Adrian Overly
AU - Saahil Singh
AU - Charlie Huang
AU - Gabriel Lopez
PY - 2026
DA - 2026/10/08
JO - PLOS One
DO - 10.1371/journal.pone.0360098
UR - https://doi.org/10.1371/journal.pone.0360098
AB - Synthetic auxotrophs are a useful means for developing genetically encoded biocontainment systems. Current methods for developing synthetic auxotrophs are complicated or expensive, limiting access and adoption of biocontainment technologies. To address this gap, we developed a simple and modular platform for creating synthetic auxotrophs based on ligand-dependent translational gating of essential genes. By inserting a theophylline-responsive riboswitch upstream of essential genes in Escherichia coli, we created strains whose viability depends on the presence of theophylline. We systematically applied this approach to 29 essential genes, obtained ligand-dependent growth phenotypes for 19 targets, and found that 18 of these essential genes yielded stringent live-die synthetic auxotrophs. These strains exhibited robust theophylline dependence with escape frequencies ranging from 1 × 10−5 to 1 × 10−6 (most below limit of detection). Our modular design allows for the rapid (<1 week), low-cost, and reliable generation of synthetic auxotrophs. This work introduces ligand-dependent translational control as a new mechanism for engineering synthetic auxotrophy and provides an accessible platform that expands the biocontainment toolkit.
SN - 1932-6203
ER -