Parallel engineering of environmental bacteria and performance over years under jungle-simulated conditions.

Engineered bacteria could perform many functions in the environment, for example, to remediate pollutants, deliver nutrients to crops or act as in-field biosensors. Model organisms can be unreliable in the field, but selecting an isolate from the thousands that naturally live there and genetically manipulating them to carry the desired function is a slow and uninformed process. Here, we demonstrate the parallel engineering of isolates from environmental samples by using the broad-host-range XPORT conjugation system (Bacillus subtilis mini-ICEBs1) to transfer a genetic payload to many isolates in parallel.

Genetically Encoding Ultrastable Virus-like Particles Encapsulating Functional DNA Nanostructures in Living Bacteria.

DNA nanotechnology is leading the field of molecular-scale device engineering, accumulating to a dazzling array of applications. However, while DNA nanostructures' function is robust under settings, their implementation in real-world conditions requires overcoming their rapid degradation and subsequent loss of function. Viruses are sophisticated supramolecular assemblies, able to protect their nucleic acid content in inhospitable biological environments.

Engineering a DNAzyme-Based Operon System for the Production of DNA Nanoscaffolds in Living Bacteria.

The ability to create nanoscaffolds within living cells using DNA has the potential to become a powerful tool in synthetic biology. However, to date, genetically encoded DNA nanostructures are limited to simple architecture due to the lack of genetic parts that can produce multiple ssDNAs in a single bacterium. Here, we develop a system that overcomes this challenge by using a single oligo gene mimicking operons.

Programmable On-Chip Artificial Cell Producing Post-Translationally Modified Ubiquitinated Protein.

In nature, intracellular microcompartments have evolved to allow the simultaneous execution of tightly regulated complex processes within a controlled environment. This architecture serves as the blueprint for the construction of a wide array of artificial cells. However, such systems are inadequate in their ability to confine and sequentially control multiple central dogma activities (transcription, translation, and post-translational modifications) resulting in a limited production of complex biomolecules.

Genetic encoding of DNA nanostructures and their self-assembly in living bacteria.

The field of DNA nanotechnology has harnessed the programmability of DNA base pairing to direct single-stranded DNAs (ssDNAs) to assemble into desired 3D structures. Here, we show the ability to express ssDNAs in Escherichia coli (32-205 nt), which can form structures in vivo or be purified for in vitro assembly. Each ssDNA is encoded by a gene that is transcribed into non-coding RNA containing a 3'-hairpin (HTBS).

Au nanoparticle/DNA rotaxane hybrid nanostructures exhibiting switchable fluorescence properties.

The preparation of a DNA rotaxane consisting of a circular nucleic acid interlocked, through hybridization, on a nucleic acid axle and stoppered by two 10-nm-sized Au nanoparticles (NPs) is described. By the tethering of 5-nm- or 15-nm-sized Au NPs on the ring, the supramolecular structure of the rotaxane is confirmed. Using nucleic acids as "fuels" and "anti-fuels", the cyclic and reversible transition of the rotaxane ring across two states is demonstrated.

Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines.

DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology.

A three-station DNA catenane rotary motor with controlled directionality.

The assembly of DNA machines represents a central effort in DNA nanotechnology. We report on the first DNA rotor system composed of a two-ring catenane. The DNA rotor ring rotates in dictated directions along a wheel, and it occupies three distinct sites.

Enzyme-free amplified detection of DNA by an autonomous ligation DNAzyme machinery.

The Zn(2+)-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal autocatalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.

pH-programmable DNA logic arrays powered by modular DNAzyme libraries.

Nature performs complex information processing circuits, such the programmed transformations of versatile stem cells into targeted functional cells. Man-made molecular circuits are, however, unable to mimic such sophisticated biomachineries. To reach these goals, it is essential to construct programmable modular components that can be triggered by environmental stimuli to perform different logic circuits.

Amplified analysis of DNA by the autonomous assembly of polymers consisting of DNAzyme wires.

A systematic study of the amplified optical detection of DNA by Mg(2+)-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10(-9) M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg(2+)-dependent DNAzyme subunits.

pH-programmable DNAzyme nanostructures.

Two engineered DNA nanostructures consisting of a nucleic acid functional hairpin and a DNA "tweezers" assembly act as pH-switchable devices for the "ON-OFF" activation/deactivation of the horseradish-peroxidase-mimicking DNAzyme.

DNA machines: bipedal walker and stepper.

The assembly of a "bipedal walker" and of a "bipedal stepper" using DNA constructs is described. These DNA machines are activated by H(+)/OH(-) and Hg(2+)/cysteine triggers. The bipedal walker is activated on a DNA template consisting of four nucleic acid footholds.

All-DNA finite-state automata with finite memory.

Biomolecular logic devices can be applied for sensing and nano-medicine. We built three DNA tweezers that are activated by the inputs H(+)/OH(-); ; nucleic acid linker/complementary antilinker to yield a 16-states finite-state automaton. The outputs of the automata are the configuration of the respective tweezers (opened or closed) determined by observing fluorescence from a fluorophore/quencher pair at the end of the arms of the tweezers.

DNA computing circuits using libraries of DNAzyme subunits.

Biological systems that are capable of performing computational operations could be of use in bioengineering and nanomedicine, and DNA and other biomolecules have already been used as active components in biocomputational circuits. There have also been demonstrations of DNA/RNA-enzyme-based automatons, logic control of gene expression, and RNA systems for processing of intracellular information. However, for biocomputational circuits to be useful for applications it will be necessary to develop a library of computing elements, to demonstrate the modular coupling of these elements, and to demonstrate that this approach is scalable.

pH-triggered switchable Mg2+-dependent DNAzymes.

The activities of Mg(2+)-dependent DNAzymes are reversibly switched by pH stimuli using the i-motif as an activating motif.

Ion-induced DNAzyme switches.

The activities of Mg(2+)-dependent DNAzymes are reversibly switched by ion stimuli using the thymine-Hg(2+)-thymine complexes or cytosine-Ag(+)-cytosine complexes.

Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst.

The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme is assembled on Au electrodes. It reveals bioelectrocatalytic properties and electrocatalyzes the reduction of H(2)O(2). The bioelectrocatalytic functions of the hemin/G-quadruplex DNAzyme are used to develop electrochemical sensors that follow the activity of glucose oxidase and biosensors for the detection of DNA or low-molecular-weight substrates (adenosine monophosphate, AMP).

Nanoengineered electrically contacted enzymes on DNA scaffolds: functional assemblies for the selective analysis of Hg2+ ions.

A DNA construct consisting of a nucleic acid template, (1), on which a nucleic acid-modified glucose oxidase (GOx), (3), was hybridized by cooperative bridging of the T-Hg(2+)-T units, and a nucleic acid-functionalized ferrocene, (5), was directly hybridized on a Au electrode. The resulting nanostructure revealed bioelectrocatalytic activities, where the ferrocene units mediated electron transfer between the redox center of the enzyme and the electrode. The bioelectrocatalytic functions of the system are regulated by the concentration of Hg(2+) ions, which controls the content of the enzyme associated with the DNA template by means of the T-Hg(2+)-T bridging units.

Electrochemically stimulated pH changes: a route to control chemical reactivity.

A bis-aniline-cross-linked Au nanoparticle (NP) composite is electrochemically prepared on a rough Pt film supported on a Au electrode. The electrochemical oxidation of the bis-aniline units to the quinoid state releases protons to the electrolyte solution, while the reduction of the quinoid bridges results in the uptake of protons from the electrolyte. By the cyclic oxidation of the bridging units (E = 0.

pH-stimulated concurrent mechanical activation of two DNA "tweezers". A "SET-RESET" logic gate system.

A DNA tweezer consisting of C-rich arms is kept in the "closed" form by hybridization of the arms with a nucleic acid cross-linker. At acidic pH (pH = 5.2), the arms are stabilized through the formation of the i-motif, C-quadruplex structures, releasing the cross-linking nucleic acid and transforming the tweezer to its "opened" state.