The epithelial mesenchymal transition (EMT) plays significant roles in the progression of cancer and fibrotic disease. Moreover, this process is reversible, resulting in mesenchymal epithelial transition (MET), which plays an important role in cancer metastasis. There is a lack of methods to trace and target EMT cells using synthetic biology circuits, which makes it difficult to study the cell fate or develop targeted treatments. In this study, we introduced responsive EMT sensing circuits, which sense the EMT using specific promoters that respond to transcription factors typical of EMT activation (EMT-TFs). The transcriptional strength of EMT-sensing promoters decreased more than 13-fold in response to the overexpression of the EMT-TF. Then, the NOT gate circuits were built by placing the tetR transcription repressor under the control of EMT sensing promoters and expressed an output signal using the constitutive CMV promoter modified with tetO sites This circuit is named EMT sensing and responding circuits .When the EMT transcription factors was present, we observed a 5.8-fold signal increase in the system. Then, we successfully distinguished mesenchymal breast cancer cells from epithelial cancer cells and repressed the proliferation of EMT tumor cells using our circuits. The EMT sensing and responding circuits are promising tools for the identification of EMT cells, which is crucial for EMT-related disease therapy and investigating the mechanisms underlying the reversible EMT process.

Synthetic biology is a broad term covering multiple scientific methodologies, technologies, and practices. Pairing biology with engineering, synbio seeks to design and build biological systems, either through improving living cells by adding in new functions, or creating new structures by combining natural and synthetic components. As with all new technologies, synthetic biology raises a number of ethical considerations. In order to understand what these issues might be, and how they relate to those covered in ethics literature on synbio, we conducted an interview study with practicing synthetic biologists affiliated with a synthetic biology centre in Australia. Scientists identified a range of ethical challenges germane to the field, including precarious employment, pressures from industry, gender inequity, and the negative effects of the hyping of synbio. These challenges differed markedly from those identified in the ethics literature, whose treatment of the harms and benefits of synbio remains largely speculative and abstract. In our discussion of the pragmatic, every day ethical issues synthetic biologists face, we illustrate how issues of waste or research integrity play pivotal roles in everything from lived experiences in the laboratory, to long-term research trajectories guiding the field. In a confirmation of the ethical relevance of our participant\'s views on the field, we argue that the subjects they raise must be included in any ethical analysis of synbio as a field.

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

Bottom-up design of biomimetic organelles has gained recent attention as a route towards understanding the transition between non-living matter and life. Despite various artificial lipid membranes being developed, the specific relations between lipid structure, composition, interfacial properties, and morphology are not currently understood. Sponge-phase droplets contain dense, nonlamellar lipid bilayer networks that capture the complexities of the endoplasmic reticulum (ER), making them ideal artificial models of such organelles. Here, we combine ultrafast two-dimensional infrared (2D IR) spectroscopy and molecular dynamics simulations to investigate the interfacial H-bond networks in sponge-phase droplets composed of glycolipid and nonionic detergents. In the sponge phase, the interfacial environments are more hydrated and water molecules confined to the nanometer-scale aqueous channels in the sponge phase exhibit dynamics that are significantly slower compared to bulk water. Surfactant configurations and microscopic phase separation play a dominant role in determining membrane curvature and slow dynamics observed in the sponge phase. The studies suggest that H-bond networks within the nanometer-scale channels are disrupted not only by confinement but also by the interactions of surfactants, which extend 1-2 nm from the bilayer surface. The results provide a molecular-level description for controlling phase and morphology in the design of synthetic lipid organelles.

The junction of bioelectrochemical systems and synthetic biology opens the door to many potentially groundbreaking technologies. When developing these possibilities, choosing the correct chassis organism can save a great deal of engineering effort and, indeed, can mean the difference between success and failure. Choosing the correct chassis for a specific application requires a knowledge of the metabolic potential of the candidate organisms, as well as a clear delineation of the traits, required in the application. In this review, we will explore the metabolic and electrochemical potential of a single genus, Marinobacter. We will cover its strengths, (salt tolerance, biofilm formation and electrochemical potential) and weaknesses (insufficient characterization of many strains and a less developed toolbox for genetic manipulation) in potential synthetic electromicrobiology applications. In doing so, we will provide a roadmap for choosing a chassis organism for bioelectrochemical systems.

Metabolic engineering for the development of microbial production strains, such as carotenoid overproducing bacteria, has a long history in industrial biotechnology. In contrast to classical strain development that mostly relies on the generation and screening of mutant libraries, rational strain development relies on the identification of a genetic target that has to be engineered in order to overcome metabolic bottlenecks facilitating the production of the desired valuable compounds. In this work, two synthetic biology approaches, namely, a CRISPRi-library and a genetically encoded biosensor, are demonstrated as tools for metabolic engineering purposes with a focus on carotenoid biosynthesis in C. glutamicum. The methods presented here gave insights into carotenoid biosynthesis and facilitated development of new metabolic engineering strategies. The use of a genetically encoded biosensor, the screening of a CRISPRi-library, and their combination can be transferred to study a wide range of organisms and target compounds.

The N-degron pathway is a branch of the ubiquitin-proteasome system where amino-terminal residues serve as degradation signals. In a synthetic biology approach, we expressed ubiquitin ligase PRT6 and ubiquitin conjugating enzyme 2 (AtUBC2) from Arabidopsis thaliana in a Saccharomyces cerevisiae strain with mutation in its endogenous N-degron pathway. The two enzymes re-constitute part of the plant N-degron pathway and were probed by monitoring the stability of co-expressed GFP-linked plant proteins starting with Arginine N-degrons. The novel assay allows for straightforward analysis, whereas in vitro interaction assays often do not allow detection of the weak binding of N-degron recognizing ubiquitin ligases to their substrates, and in planta testing is usually complex and time-consuming.

The epigenetic marker 5-methylcytosine (5mC) is an important factor in DNA modification and epigenetics. It can be modified through a three-step oxidation performed by ten-eleven-translocation (TET) enzymes and we have previously reported that the iron(IV)-oxo complex [Fe(O)(Py5 Me2 H)]2+ (1) can oxidize 5mC. Here, we report the reactivity of this iron(IV)-oxo complex towards a wider scope of methylated cytosine and uracil derivatives relevant for synthetic DNA applications, such as 1-methylcytosine (1mC), 5-methyl-iso-cytosine (5miC) and thymine (T/5mU). The observed kinetic parameters are corroborated by calculation of the C-H bond energies at the reactive sites which was found to be an efficient tool for reaction rate prediction of 1 towards methylated DNA bases. We identified oxidation products of methylated cytosine derivatives using HPLC-MS and GC-MS. Thereby, we shed light on the impact of the methyl group position and resulting C-H bond dissociation energies on reactivity towards TET-like oxidation.

The goal of achieving enhanced diagnosis and continuous monitoring of human health has led to a vibrant, dynamic and well-funded field of research in medical sensing and biosensor technologies. The field has many sub-disciplines which focus on different aspects of sensor science; engaging engineers, chemists, biochemists and clinicians, often in interdisciplinary teams. The trends which dominate include the efforts to develop effective point of care tests and implantable/wearable technologies for early diagnosis and continuous monitoring. This review will outline the current state of the art in a number of relevant fields, including device engineering, chemistry, nanoscience and biomolecular detection, and suggest how these advances might be employed to develop effective systems for measuring physiology, detecting infection and monitoring biomarker status in wild animals. Special consideration is also given to the emerging threat of antimicrobial resistance and in the light of the current SARS-CoV-2 outbreak, zoonotic infections. Both of these areas involve significant crossover between animal and human health and are therefore well placed to seed technological developments with applicability to both human and animal health and, more generally, the reviewed technologies have significant potential to find use in the measurement of physiology in wild animals. This article is part of the theme issue \'Measuring physiology in free-living animals (Part II)\'.

The non-natural ethynylmethylpyridone C-nucleoside (W), a thymidine (T) analogue that can be incorporated in oligonucleotides by automated synthesis, has recently been reported to form a high fidelity base pair with adenosine (A) and to be well accommodated in B-DNA duplexes. The enhanced binding affinity for A of W, as compared to T, makes it an ideal modification for biotechnological applications, such as efficient probe hybridization for the parallel detection of multiple DNA strands. In order to complement the experimental study and rationalize the impact of the non-natural W nucleoside on the structure, stability and dynamics of DNA structures, we performed quantum mechanics (QM) calculations along with molecular dynamics (MD) simulations. Consistently with the experimental study, our QM calculations show that the A:W base pair has an increased stability as compared to the natural A:T pair, due to an additional CH-π interaction. Furthermore, we show that mispairing between W and guanine (G) causes a distortion in the planarity of the base pair, thus explaining the destabilization of DNA duplexes featuring a G:W pair. MD simulations show that incorporation of single or multiple consecutive A:W pairs in DNA duplexes causes minor changes to the intra- and inter-base geometrical parameters, while a moderate widening/shrinking of the major/minor groove of the duplexes is observed. QM calculations applied to selected stacks from the MD simulations also show an increased stacking energy for W, over T, with the neighboring bases.