Bioindustrial manufacturing readiness levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes.

Readiness level (RL) frameworks such as technology readiness levels and manufacturing readiness levels describe the status of a technology/manufacturing process on its journey from initial conception to commercial deployment. More importantly, they provide a roadmap to guide technology development and scale-up from a ''totality of system'' approach. Commercialization risks associated with too narrowly focused R&D efforts are mitigated. RLs are defined abstractly so that they can apply to diverse industries and technology sectors. However, differences between technology sectors make necessary the definition of sector specific RL frameworks. Here, we describe bioindustrial manufacturing readiness levels (BioMRLs), a classification system specific to bioindustrial manufacturing. BioMRLs will give program managers, investors, scientists, and engineers a shared vocabulary for prioritizing goals and assessing risks in the development and commercialization of a bioindustrial manufacturing process.

Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.

Agricultural Crop Security: Exploring US Federal Readiness and Response Capabilities.

A large-scale agroterrorism attack on the United States would likely have severe economic and social consequences. In particular, the destruction of crops with pests or pathogens could cause substantial damage to food, economic, and social stability, with relatively little health risk to the perpetrators. The tools of engineering biology could enable a well-trained, nefarious actor to amplify their desired impacts through the development of disease-intensifying traits. In the United States, plant health emergencies are handled first at the local and state levels, then escalated to include the support and leadership of the US Department of Agriculture (USDA) and other federal agencies. We used publicly available documents and interviews across government, academia, and industry to explore the strategic and tactical approaches of the US federal government to detect and respond to plant agroterrorism. In this article, we discuss the agroterrorism preparedness and response capabilities at 3 levels of federal response: (1) within the Plant Protection and Quarantine program of the Animal and Plant Health Inspection Service at the USDA, (2) between USDA components, and (3) between federal agencies. We outline the approaches currently taken and identify opportunities to strengthen these approaches.

Guiding Ethical Principles in Engineering Biology Research.

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development.

Author Correction: Building a global alliance of biofoundries.

The original version of this Comment contained errors in the legend of Figure 2, in which the locations of the fifteenth and sixteenth GBA members were incorrectly given as '(15) Australian Genome Foundry, Macquarie University; (16) Australian Foundry for Advanced Biomanufacturing, University of Queensland.'. The correct version replaces this with '(15) Australian Foundry for Advanced Biomanufacturing (AusFAB), University of Queensland and (16) Australian Genome Foundry, Macquarie University'. This has been corrected in both the PDF and HTML versions of the Comment.

YouTube resources for synthetic biology education.

Online video resources have increasingly become a common way to effectively share scientific research ideas and engage viewers at many levels of interest or expertise. While synthetic biology is a comparatively young field, it has accumulated online videos across a spectrum of content and technical depth. Such video content can be used to introduce viewers to synthetic biology, supplement college course content, teach new lab skills and entertain. Here, we compile online videos concerning synthetic biology into public YouTube playlists tailored for six different, though potentially overlapping, audiences: those wanting an introduction to synthetic biology, those wanting to get quick overviews of specific topics within synthetic biology, those wanting teaching or public lectures, those wanting more technical research lectures, those wanting to learn lab protocols and those interested in the International Genetically Engineered Machine competition.

Industrialization of Biology.

The advancement of synthetic biology over the past decade has contributed substantially to the growing bioeconomy. A recent report by the National Academies highlighted several areas of advancement that will be needed for further expansion of industrial biotechnology, including new focuses on design, feedstocks, processing, organism development, and tools for testing and measurement; more particularly, a focus on expanded chassis and end-to-end design in an effort to move beyond the use of E. coli and S. cerivisiea to organisms better suited to fermentation and production; second, continued efforts in systems biology and high-throughput screening with a focus on more rapid techniques that will provide the needed information for moving to larger scale; and finally, work to accelerate the building of a holacratic community with collaboration and engagement between the relevant government agencies, industry, academia, and the public.

Self-assembly of a [2]pseudorota[3]catenane in water.

A donor-acceptor [3]catenane incorporating two cyclobis(paraquat-p-phenylene) rings linked together by a dinaphtho[50]crown-14 macrocycle possesses a π-electron-deficient pocket. Contrary to expectation, negligible binding of a hexaethylene glycol chain interrupted in its midriff by a π-electron-rich 1,5-dioxynaphthalene unit was observed in acetonitrile. However, a fortuitous solid-state superstructure of the expected 1:1 complex revealed its inability to embrace any stabilizing [C-H···O] interactions between the clearly unwelcome guest and the host reluctantly accommodating it. By contrast, in aqueous solution, the 1:1 complex becomes very stable thanks to the intervention of hydrophobic bonding.

Positive cooperativity in the template-directed synthesis of monodisperse macromolecules.

Two series of oligorotaxanes R and R' that contain -CH(2)NH(2)(+)CH(2)- recognition sites in their dumbbell components have been synthesized employing template-directed protocols. [24]Crown-8 rings self-assemble by a clipping strategy around each and every recognition site using equimolar amounts of 2,6-pyridinedicarboxaldehyde and tetraethyleneglycol bis(2-aminophenyl) ether to efficiently provide up to a [20]rotaxane. In the R series, the -NH(2)(+)- recognition sites are separated by trismethylene bridges, whereas in the R' series the spacers are p-phenylene linkers. The underpinning idea here is that in the former series, the recognition sites are strategically positioned 3.5 Å apart from one another so as to facilitate efficient [π···π] stacking between the aromatic residues in contiguous rings in the rotaxanes and consequently, a discrete rigid and rod-like conformation is realized; these noncovalent interactions are absent in the latter series rendering them conformationally flexible/nondiscrete. Although in the R' series, the [3]-, [4]-, [8]-, and [12]rotaxanes were isolated after reaction times of <5-30 min in yields of 72-85%, in the R series, the [3]-, [4]-, [5]-, [8]-, [12]-, [16]-, and [20]rotaxanes were isolated in <5 min to 14 h in 88-98% yields. It follows that while in the R' series the higher order oligorotaxanes are formed in lower yields more rapidly, in the R series, the higher order oligorotaxanes are formed in higher yields more slowly. In the R series, the high percentage yields are sustained throughout, despite the fact that up to 39 components are participating in the template-directed self-assembly process. Simple arithmetic reveals that the conversion efficiency for each imine bond formation peaks at 99.9% in the R series and 99.3% in the R' series. This maintenance of reaction efficiency in the R series can be ascribed to positive cooperativity, that is, when one ring is formed it aids and abets the formation of subsequent rings presumably because of stabilizing extended [π···π] stacking interactions between the arene units. Experiments have been performed wherein the dumbbell is starved of the macrocyclic components, and up to five times more of the fully saturated rotaxane is formed than is predicted based on a purely statistical outcome, providing a clear indication that positive cooperativity is operative. Moreover, it would appear that as the R series is traversed from the [3]- to the [4]- to the [5]rotaxane, the cooperativity becomes increasingly positive. This kind of cooperative behavior is not observed for the analogous oligorotaxanes in the R' series. The conventional bevy of analytical techniques (e.g., HR-MS (ESI) and both (1)H and (13)C NMR spectroscopy) help establish the fact that all the oligorotaxanes are pure and monodisperse. Evidence of efficient [π···π] stacking between contiguous arene units in the rings in the R series is revealed by (1)H NMR spectroscopy. Ion-mobility mass spectrometry performed on the R and R' series yielded the collisional cross sections (CCSs), confirming the rigidity of the R oligorotaxanes and the flexibility of the R' ones. The extended [π···π] stacking interactions are found to be present in the solid-state structures of the [3]- and [4]rotaxanes in the R series and also on the basis of molecular mechanics calculations performed on the entire series of oligomers. The collective data presented herein supports our original design in that the extended [π···π] stacking between contiguous arene units in the rings of the R series of oligorotaxanes facilitate an essentially rigid rod-like conformation with evidence that positive cooperativity improves the efficiency of their formation. This situation stands in sharp contrast to the conformationally flexible R' series where the oligorotaxanes form with no cooperativity.

Donor-acceptor ring-in-ring complexes.

The self-assembly of three donor-acceptor ring-in-ring complexes, prepared from the π-electron-deficient tetracationic cyclophane, cyclobis(paraquat-4,4'-biphenylene), and three large π-electron-rich crown ethers (each 50-membered rings) containing dioxynaphthalene (DNP) and tetrathiafulvalene (TTF) units in pairs (DNP/DNP, DNP/TTF and TTF/TTF), is reported. (1)H NMR spectroscopic analyses are indicative of the formation of 1:1 complexes in CD(3)CN, whilst the charge-transfer interactions between the DNP and TTF units of the crown ethers and the tetracationic cyclophane have permitted the measurement of binding constants of up to 4×10(3) M(-1) in CH(3)CN to be made using UV/Vis spectroscopy. Ring-in-ring complexes are proposed as intermediates in the stepwise synthesis of molecular Borromean rings (BRs) comprised of three different rings. With the particular choice of crown ethers, the 1:1 complexes have polyether loops that protrude from the donor-acceptor recognition point above and below the mean plane of the tetracationic cyclophane, which, ideally, could conceivably bind dialkylammonium centers present in a third ring. X-ray crystallographic analyses of the solid-state superstructures of two of the three 1:1 complexes reveal, however, the presence of prodigious CH···O interactions between the polyether loops of the crown ethers and the rims of the cyclophane, no doubt stabilizing the complexes, but, at the same time, masking their potential recognition sites from further interactions that are essential to the subsequent emergence of the third ring. The solid-state superstructure of one of the crown ethers binding two dibenzylammonium ions provides some insight into the design requirements for the next generation of these systems; longer polyether loops may be required to allow optimal interactions between all components. It has become clear during a pursuit of the stepwise synthesis of the molecular BRs that, when designing complex mechanically interlocked molecules utilizing multiple recognition sites, the unsullied orthogonality of the recognition motifs is of the utmost importance.

Donor-acceptor molecular figures-of-eight.

The intermolecular template-directed synthesis, separation and characterisation of two constitutional isomers that are self-complexing donor-acceptor [1]rotaxanes has been achieved by click chemistry, starting from a π-electron deficient tetracationic cyclophane containing two azide functions and a π-electron rich 1,5-dioxynaphthalene-containing polyether chain terminated by propargyl groups.

Mechanically interlocked mechanophores by living-radical polymerization from rotaxane initiators.

Two [2]rotaxane initiators for single-electron-transfer living-radical-polymerization were synthesized and used for the controlled polymerization of methyl acrylate. The mechanically interlocked polymers exhibited distinct responses to mechanical activation by ultrasound. Monitoring the fate of the rotaxanes' charge transfer absorption bands provides evidence for preferential mechanical degradation of a midsection rotaxane unit as compared to a terminal rotaxane entity as a consequence of mechanical forces accumulating in the central region of the polymer chain.

Mechanically stabilized tetrathiafulvalene radical dimers.

Two donor-acceptor [3]catenanes-composed of a tetracationic molecular square, cyclobis(paraquat-4,4'-biphenylene), as the π-electron deficient ring and either two tetrathiafulvalene (TTF) and 1,5-dioxynaphthalene (DNP) containing macrocycles or two TTF-butadiyne-containing macrocycles as the π-electron rich components-have been investigated in order to study their ability to form TTF radical dimers. It has been proven that the mechanically interlocked nature of the [3]catenanes facilitates the formation of the TTF radical dimers under redox control, allowing an investigation to be performed on these intermolecular interactions in a so-called "molecular flask" under ambient conditions in considerable detail. In addition, it has also been shown that the stability of the TTF radical-cation dimers can be tuned by varying the secondary binding motifs in the [3]catenanes. By replacing the DNP station with a butadiyne group, the distribution of the TTF radical-cation dimer can be changed from 60% to 100%. These findings have been established by several techniques including cyclic voltammetry, spectroelectrochemistry and UV-vis-NIR and EPR spectroscopies, as well as with X-ray diffraction analysis which has provided a range of solid-state crystal structures. The experimental data are also supported by high-level DFT calculations. The results contribute significantly to our fundamental understanding of the interactions within the TTF radical dimers.

Donor-acceptor oligorotaxanes made to order.

Five donor-acceptor oligorotaxanes made up of dumbbells composed of tetraethylene glycol chains, interspersed with three and five 1,5-dioxynaphthalene units, and terminated by 2,6-diisopropylphenoxy stoppers, have been prepared by the threading of discrete numbers of cyclobis(paraquat-p-phenylene) rings, followed by a kinetically controlled stoppering protocol that relies on click chemistry. The well-known copper(I)-catalyzed alkyne-azide cycloaddition between azide functions placed at the ends of the polyether chains and alkyne-bearing stopper precursors was employed during the final kinetically controlled template-directed synthesis of the five oligorotaxanes, which were characterized subsequently by (1)H NMR spectroscopy at low temperature (233 K) in deuterated acetonitrile. The secondary structures, as well as the conformations, of the five oligorotaxanes were unraveled by spectroscopic comparison with the dumbbell and ring components. By focusing attention on the changes in chemical shifts of some key probe protons, obtained from a wide range of low-temperature spectra, a picture emerges of a high degree of folding within the thread protons of the dumbbells of four of the five oligorotaxanes-the fifth oligorotaxane represents a control compound in effect-brought about by a combination of C-H···O and π-π stacking interactions between the π-electron-deficient bipyridinium units in the rings and the π-electron-rich 1,5-dioxynaphthalene units and polyether chains in the dumbbells. The secondary structures of a foldamer-like nature have received further support from a solid-state superstructure of a related [3]pseudorotaxane and density functional calculations performed thereon.

A multistate switchable [3]rotacatenane.

Rotacatenanes are exotic molecular compounds that can be visualized as a unique combination of a [2]catenane and a [2]rotaxane, thereby combining both the circumrotation of the ring component (rotary motion) and the shuttling of the dumbbell component (translational motion) in one structure. Herein, we describe a strategy for the synthesis of a new switchable [3]rotacatenane and the investigation of its switching properties, which rely on the formation of tetrathiafulvalene (TTF) radical π-dimer interactions-namely, the mixed-valence state (TTF(2) )(+.) and the radical-cation dimer state (TTF(+.) )(2) -under ambient conditions. A template-directed approach, based on donor-acceptor interactions, has been developed, resulting in an improved yield of the key precursor [2]catenane, prior to rotacatenation. The nature of the binding between the [2]catenane and selected π-electron-rich templates has been elucidated by using X-ray crystallography and UV/Vis spectroscopy as well as isothermal titration microcalorimetry. The multistate switching mechanism of the [3]rotacatenane has been demonstrated by cyclic voltammetry and EPR spectroscopy. Most notably, the radical-cation dimer state (TTF(+.) )(2) has been shown to enter into an equilibrium by forming the co-conformation in which the two 1,5-dioxynaphthalene (DNP) units co-occupy the cavity of tetracationic cyclophane, thus enforcing the separation of TTF radical-cation dimer (TTF(+.) )(2) . The population ratio of this equilibrium state was found to be 1:1. We believe that this research demonstrates the power of constructing complex molecular machines using template-directed protocols, enabling us to make the transition from simple molecular switches to their multistate variants for enhancing information storage in molecular electronic devices.

Radically enhanced molecular recognition.

The tendency for viologen radical cations to dimerize has been harnessed to establish a recognition motif based on their ability to form extremely strong inclusion complexes with cyclobis(paraquat-p-phenylene) in its diradical dicationic redox state. This previously unreported complex involving three bipyridinium cation radicals increases the versatility of host-guest chemistry, extending its practice beyond the traditional reliance on neutral and charged guests and hosts. In particular, transporting the concept of radical dimerization into the field of mechanically interlocked molecules introduces a higher level of control within molecular switches and machines. Herein, we report that bistable and tristable [2]rotaxanes can be switched by altering electrochemical potentials. In a tristable [2]rotaxane composed of a cyclobis(paraquat-p-phenylene) ring and a dumbbell with tetrathiafulvalene, dioxynaphthalene and bipyridinium recognition sites, the position of the ring can be switched. On oxidation, it moves from the tetrathiafulvalene to the dioxynaphthalene, and on reduction, to the bipyridinium radical cation, provided the ring is also reduced simultaneously to the diradical dication.

Highly stable tetrathiafulvalene radical dimers in [3]catenanes.

Two [3]catenane 'molecular flasks' have been designed to create stabilized, redox-controlled tetrathiafulvalene (TTF) dimers, enabling their spectrophotometric and structural properties to be probed in detail. The mechanically interlocked framework of the [3]catenanes creates the ideal arrangement and ultrahigh local concentration for the encircled TTF units to form stable dimers associated with their discrete oxidation states. These dimerization events represent an affinity umpolung, wherein the inversion in electronic affinity replaces the traditional TTF-bipyridinium interaction, which is over-ridden by stabilizing mixed-valence (TTF)2•+ and radical-cation (TTF•+)2 states inside the 'molecular flasks.' The experimental data, collected in the solid state as well as in solution under ambient conditions, together with supporting quantum mechanical calculations, are consistent with the formation of stabilized paramagnetic mixed-valence dimers, and then diamagnetic radical-cation dimers following subsequent one-electron oxidations of the [3]catenanes.

Directed self-assembly of a ring-in-ring complex.

A strategic modification to the corner ligands in Pd(II)-containing, electron-poor cyclophanes has profound repercussions for their assemblies with electron-rich aromatic crown ethers in both the solid and solution states; the formation of ring-in-ring complexes can override competing [3]catenane production on masking the hydrogen bond donor capabilities of the corner ligands.