Metaphors as design tools for microbial consortia: An analysis of recent peer-reviewed literature.

Single engineered microbial species cannot always conduct complex transformations, while complex, incompletely defined microbial consortia have heretofore been suited to a limited range of tasks. As biodesigners bridge this gap with intentionally designed microbial communities, they will, intentionally or otherwise, build communities that embody particular ideas about what microbial communities can and should be. Here, we suggest that metaphors-ideas about what microbial communities are like-are therefore important tools for designing synthetic consortia-based bioreactors. We identify a range of metaphors currently employed in peer-reviewed microbiome research articles, characterizing each through its potential structural implications and distinctive imagery. We present this metaphor catalogue in the interest of, first, making metaphors visible as design choices, second, enabling deliberate experimentation with them towards expanding the potential design space of the field, and third, encouraging reflection on the goals and values they embed.

Reconfiguring the Challenge of Biological Complexity as a Resource for Biodesign.

Biological complexity is widely seen as the central, intractable challenge of engineering biology. Yet this challenge has been constructed through the field's dominant metaphors. Alternative ways of thinking-latent in progressive experimental approaches, but rarely articulated as such-could instead position complexity as engineering biology's greatest resource. We outline how assumptions about engineered microorganisms have been built into the field, carried by entrenched metaphors, even as contemporary methods move beyond them. We suggest that alternative metaphors would better align engineering biology's conceptual infrastructure with the field's move away from conventionally engineering-inspired methods toward biology-centric ones. Innovating new conceptual frameworks would also enable better aligning scientific work with higher-level conversations about that work. Such innovation-thinking about how engineering microbes might be more like user-centered design than like programming a computer or building a car-could highlight complexity as a resource to leverage, not a problem to erase or negate.

Using Cartesian Doubt To Build a Sequencing-Based View of Microbiology.

The technological leap of DNA sequencing generated a tension between modern metagenomics and historical microbiology. We are forcibly harmonizing the output of a modern tool with centuries of experimental knowledge derived from culture-based microbiology. As a thought experiment, we borrow the notion of Cartesian doubt from philosopher Rene Descartes, who used doubt to build a philosophical framework from his incorrigible statement that "I think therefore I am." We aim to cast away preconceived notions and conceptualize microorganisms through the lens of metagenomic sequencing alone. Specifically, we propose funding and building analysis and engineering methods that neither search for nor rely on the assumption of independent genomes bound by lipid barriers containing discrete functional roles and taxonomies. We propose that a view of microbial communities based in sequencing will engender novel insights into metagenomic structure and may capture functional biology not reflected within the current paradigm.

Words Are Essential, but Underexamined, Research Tools for Microbes and Microbiomes.

Language constitutes an essential set of scientific construction tools, not only for communicating knowledge, but for conceptualizing the world. Metaphors in particular, as conventions that guide and reproduce analogical reasoning, merit attention that they largely do not receive. My research addresses this deficit by examining how metaphors for handling microbes shape possibilities for working with yeast and bacteria in synthetic biology, microbiome research, and other fields that reconfigure what microbes can be. Though poised to reexamine assumptions, these fields routinely rest on metaphors and other language tools that quietly embed ways of thinking that may work against wider aims-for example, imagining bacteria as imperfect machines that should therefore be rendered increasingly passive and controllable. Researchers, therefore, need to examine how language tools structure their observations and expectations so that the tools they choose are appropriate for the work they want to do.

Crossing Kingdoms: How Can Art Open Up New Ways of Thinking About Science?

"Crossing Kingdoms" is an artist-led experiment in the biological fusion of mammalian and yeast cells and the cultural discussions of these phenomena. We present this collaboration as an experiment in responsible research and innovation (RRI), an institutionalized format for ensuring that researchers reflect on the wider social dimensions of their work. Our methods challenged us as researchers to reflect on interdisciplinary collaboration and the possibility of innovating in biology for artistic purposes, challenged audiences to reflect on biological boundaries, and challenged both groups to reflect on what it means to be responsible in science. We conclude that our experiment in RRI was successful because we have asked unexpected questions-a contrast to RRI implemented as a standard protocol. Our experiment has implications for biologists and artists pursuing interdisciplinary collaborations with each other and for researchers thinking about implementing RRI as more than a box-ticking exercise.

Models for DNA Design Tools: The Trouble with Metaphors Is That They Don't Go Away.

Synthetic biology relies heavily on DNA design tools to enable manipulation of DNA in silico. Existing tools, however, are falling short of enabling aspirations for the field that emphasize efficient, automated design pipelines. We review existing DNA design tools, identify underlying similarities in how they model correlations between DNA structure and function, and suggest that iterating the existing model is unlikely to overcome limitations in matching software applications to design aspirations. The current model is predicated on metaphors conceptualizing DNA as linear text, accounting for relatively little of the known complexity of DNA function. New models that can account for more of that complexity and thus enable more ambitious DNA design goals are likely to call for new underlying metaphors-a need that may be addressed by rethinking DNA in terms of human rather than computer languages.

Designing with living systems in the synthetic yeast project.

Synthetic biology is challenged by the complexity and the unpredictability of living systems. While one response to this complexity involves simplifying cells to create more fully specified systems, another approach utilizes directed evolution, releasing some control and using unpredictable change to achieve design goals. Here we discuss SCRaMbLE, employed in the synthetic yeast project, as an example of synthetic biology design through working with living systems. SCRaMbLE is a designed tool without being a design tool, harnessing the activities of the yeast rather than relying entirely on scientists' deliberate choices. We suggest that directed evolution at the level of the whole organism allows scientists and microorganisms to "collaborate" to achieve design goals, suggesting new directions for synthetic biology.

Who are the users of synthetic DNA? Using metaphors to activate microorganisms at the center of synthetic biology.

Synthetic biology, a multidisciplinary field involving designing and building with DNA, often designs and builds in microorganisms. The role of these microorganisms tends to be understood through metaphors making the microbial cell like a machine and emphasizing its passivity: cells are described as platforms, chassis, and computers. Here, I point to the efficacy of such metaphors in enacting the microorganism as a particular kind of (non-)participant in the research process, and I suggest the utility of employing metaphors that make microorganisms a different kind of thing-active participants, contributors, and even collaborators in scientific research. This suggestion is worth making, I argue, because enabling the activity of the microorganism generates opportunities for learning from microorganisms in ways that may help explain currently unexplained phenomena in synthetic biology and suggest new experimental directions. Moreover, "activating the microorganism" reorients relationships between human scientists and nonhuman experimental participants away from control over nonhuman creatures and toward respect for and listening to them, generating conditions of possibility for exploring what responsible research means when humans try to be responsible toward and even with creatures across species boundaries.

Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage.

We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.

The bacterium, nontypeable Haemophilus influenzae, enhances host antiviral response by inducing Toll-like receptor 7 expression: evidence for negative regulation of host anti-viral response by CYLD.

The incidence of mixed viral/bacterial infections has increased recently because of the dramatic increase in antibiotic-resistant strains, the emergence of new pathogens, and the resurgence of old ones. Despite the relatively well-known role of viruses in enhancing bacterial infections, the impact of bacterial infections on viral infections remains unknown. In this study, we provide direct evidence that nontypeable Haemophilus influenzae (NTHi), a major respiratory bacterial pathogen, augments the host antiviral response by up-regulating epithelial Toll-like receptor 7 (TLR7) expression in vitro and in vivo. Moreover, NTHi induces TLR7 expression via a TLR2-MyD88-IRAK-TRAF6-IKK-NF-kappaB-dependent signaling pathway. Interestingly, CYLD, a novel deubiquitinase, acts as a negative regulator of TLR7 induction by NTHi. Our study thus provides new insights into a novel role for bacterial infection in enhancing host antiviral response and further identifies CYLD for the first time as a critical negative regulator of host antiviral response.