Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels, and chemicals1. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems2,3, which include bioremediation4 and probiotics5. Although safeguards have been designed to control cell growth by essential gene regulation6, inducible toxin switches7, and engineered auxotrophies8, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations9,10. Here, we describe the construction of a series of genomically recoded organisms (GROs)11 whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase (aaRS) pair into the chromosome of a GRO that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering (MAGE)12, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harboring 3 TAG codons in conserved functional residues13 of MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ∼1011 cells on solid media for seven days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches2,3,6-10. We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternate genetic codes that impart genetic isolation by impeding horizontal gene transfer11 and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

A major goal of nanotechnology and bioengineering is to build artificial nanomachines capable of generating specific membrane curvatures on demand. Inspired by natural membrane-deforming proteins, we designed DNA-origami curls that polymerize into nanosprings and show their efficacy in vesicle deformation. DNA-coated membrane tubules emerge from spherical vesicles when DNA-origami polymerization or high membrane-surface coverage occurs. Unlike many previous methods, the DNA self-assembly-mediated membrane tubulation eliminates the need for detergents or top-down manipulation. The DNA-origami design and deformation conditions have substantial influence on the tubulation efficiency and tube morphology, underscoring the intricate interplay between lipid bilayers and vesicle-deforming DNA structures.

Lipid and DNA are abundant biomolecules with critical functions in cells. The water-insoluble, amphipathic lipid molecules are best known for their roles in energy storage (e.g. as triglyceride), signaling (e.g. as sphingolipid), and compartmentalization (e.g. by forming membrane-enclosed bodies). The soluble, highly negatively charged DNA, which stores cells' genetic information, has proven to be an excellent material for constructing programmable nanostructures in vitro thanks to its self-assembling capabilities. These two seemingly distant molecules make contact within cell nuclei, often via lipidated proteins, with proposed functions of modulating chromatin structures. Carefully formulated lipid/DNA complexes are promising reagents for gene therapy. The past few years saw an emerging research field of interfacing DNA nanostructures with lipid membranes, with an overarching goal of generating DNA/lipid hybrid materials that possess novel and controllable structure, dynamics, and function. An arsenal of DNA-based tools has been created to coat, mold, deform, and penetrate lipid bilayers, affording us the ability to manipulate membranes with nanoscopic precision. These membrane engineering methods not only enable quantitative biophysical studies, but also open new opportunities in synthetic biology (e.g. artificial cells) and therapeutics (e.g. drug delivery).

DNA nanotechnology provides an avenue for the construction of rationally designed artificial assemblages with well-defined and tunable architectures. Shaped to mimic natural membrane-deforming proteins and equipped with membrane anchoring molecules, curved DNA nanostructures can reproduce subcellular membrane remodeling events such as vesicle tubulation in vitro. To systematically analyze how structural stiffness and membrane affinity of DNA nanostructures affect the membrane remodeling outcome, here we build DNA-origami curls with varying thickness and amphipathic peptide density, and have them polymerize into nanosprings on the surface of liposomes. We find that modestly reducing rigidity and maximizing the number of membrane anchors not only promote membrane binding and remodeling but also lead to the formation of lipid tubules with better defined diameters, highlighting the ability of programmable DNA-based constructs to controllably deform the membrane.

Viruses build nucleotide Z, identified in meteorites, replacing adenine in DNA genomes