Confronting risks of mirror life.

Broad discussion is needed to chart a path forward.

Genome-wide transcription response of to heat shock and medically relevant glucose levels.

Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. We quantified genome-wide changes in gene expression levels for one abundant skin commensal, , in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature.

Engineering tRNA abundances for synthetic cellular systems.

Routinizing the engineering of synthetic cells requires specifying beforehand how many of each molecule are needed. Physics-based tools for estimating desired molecular abundances in whole-cell synthetic biology are missing. Here, we use a colloidal dynamics simulator to make predictions for how tRNA abundances impact protein synthesis rates.

Colloidal Physics Modeling Reveals How Per-Ribosome Productivity Increases with Growth Rate in Escherichia coli.

Faster-growing cells must synthesize proteins more quickly. Increased ribosome abundance only partly accounts for increases in total protein synthesis rates. The productivity of individual ribosomes must increase too, almost doubling by an unknown mechanism.

Direct and indirect impacts of synthetic biology on biodiversity conservation.

The world's biodiversity is in crisis. Synthetic biology has the potential to transform biodiversity conservation, both directly and indirectly, in ways that are negative and positive. However, applying these biotechnology tools to environmental questions is fraught with uncertainty and could harm cultures, rights, livelihoods, and nature.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue.

Permutational analysis of regulatory elements.

Gene expression in is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements-enhancers (upstream activating sequences), core promoters, 5' untranslated regions (5' UTRs) and transcription terminators/3' untranslated regions (3' UTRs). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remain under explored.

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2.

Growing a circular economy with fungal biotechnology: a white paper.

Fungi have the ability to transform organic materials into a rich and diverse set of useful products and provide distinct opportunities for tackling the urgent challenges before all humans. Fungal biotechnology can advance the transition from our petroleum-based economy into a bio-based circular economy and has the ability to sustainably produce resilient sources of food, feed, chemicals, fuels, textiles, and materials for construction, automotive and transportation industries, for furniture and beyond. Fungal biotechnology offers solutions for securing, stabilizing and enhancing the food supply for a growing human population, while simultaneously lowering greenhouse gas emissions.

Enabling community-based metrology for wood-degrading fungi.

Background: Lignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture.

Definitive demonstration by synthesis of genome annotation completeness.

We develop a method for completing the genetics of natural living systems by which the absence of expected future discoveries can be established. We demonstrate the method using bacteriophage øX174, the first DNA genome to be sequenced. Like many well-studied natural organisms, closely related genome sequences are available-23 genomes related to øX174.

Fail-safe genetic codes designed to intrinsically contain engineered organisms.

One challenge in engineering organisms is taking responsibility for their behavior over many generations. Spontaneous mutations arising before or during use can impact heterologous genetic functions, disrupt system integration, or change organism phenotype. Here, we propose restructuring the genetic code itself such that point mutations in protein-coding sequences are selected against.

Author Correction: Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Opening options for material transfer.

Unlabelled: The Open Material Transfer Agreement is a material-transfer agreement that enables broader sharing and use of biological materials by biotechnology practitioners working within the practical realities of technology transfer.

Supplementary Information: The online version of this article (doi:10.1038/nbt.

Reconstruction of cysteine biosynthesis using engineered cysteine-free enzymes.

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids.

Measurements of translation initiation from all 64 codons in E. coli.

Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the 'start codons' for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons.

Artificial Symmetry-Breaking for Morphogenetic Engineering Bacterial Colonies.

Morphogenetic engineering is an emerging field that explores the design and implementation of self-organized patterns, morphologies, and architectures in systems composed of multiple agents such as cells and swarm robots. Synthetic biology, on the other hand, aims to develop tools and formalisms that increase reproducibility, tractability, and efficiency in the engineering of biological systems. We seek to apply synthetic biology approaches to the engineering of morphologies in multicellular systems.

When Wavelengths Collide: Bias in Cell Abundance Measurements Due to Expressed Fluorescent Proteins.

The abundance of bacteria in liquid culture is commonly inferred by measuring optical density at 600 nm. Red fluorescent proteins (RFPs) can strongly absorb light at 600 nm. Increasing RFP expression can falsely inflate apparent cell density and lead to underestimations of mean per-cell fluorescence by up to 10%.

Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates.

Whole-cell biosensors have several advantages for the detection of biological substances and have proven to be useful analytical tools. However, several hurdles have limited whole-cell biosensor application in the clinic, primarily their unreliable operation in complex media and low signal-to-noise ratio. We report that bacterial biosensors with genetically encoded digital amplifying genetic switches can detect clinically relevant biomarkers in human urine and serum.

The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology.

The re-use of previously validated designs is critical to the evolution of synthetic biology from a research discipline to an engineering practice. Here we describe the Synthetic Biology Open Language (SBOL), a proposed data standard for exchanging designs within the synthetic biology community. SBOL represents synthetic biology designs in a community-driven, formalized format for exchange between software tools, research groups and commercial service providers.

One-step cloning and chromosomal integration of DNA.

We describe "clonetegration", a method for integrating DNA into prokaryotic chromosomes that approaches the simplicity of cloning DNA within extrachromosomal vectors. Compared to existing techniques, clonetegration drastically decreases the time and effort needed for integration of single or multiple DNA fragments. Additionally, clonetegration facilitates cloning and expression of genetic elements that are impossible to propagate within typical multicopy plasmids.