Genetically programmed synthetic cells for thermo-responsive protein synthesis and cargo release.

Synthetic cells containing genetic programs and protein expression machinery are increasingly recognized as powerful counterparts to engineered living cells in the context of biotechnology, therapeutics and cellular modelling. So far, genetic regulation of synthetic cell activity has been largely confined to chemical stimuli; to unlock their potential in applied settings, engineering stimuli-responsive synthetic cells under genetic regulation is imperative. Here we report the development of temperature-sensitive synthetic cells that control protein production by exploiting heat-responsive mRNA elements. This is achieved by combining RNA thermometer technology, cell-free protein expression and vesicle-based synthetic cell design to create cell-sized capsules able to initiate synthesis of both soluble proteins and membrane proteins at defined temperatures. We show that the latter allows for temperature-controlled cargo release phenomena with potential implications for biomedicine. Platforms like the one presented here can pave the way for customizable, genetically programmed synthetic cells under thermal control to be used in biotechnology.

Burden-driven feedback control of gene expression.

Cells use feedback regulation to ensure robust growth despite fluctuating demands for resources and differing environmental conditions. However, the expression of foreign proteins from engineered constructs is an unnatural burden that cells are not adapted for. Here we combined RNA-seq with an in vivo assay to identify the major transcriptional changes that occur in Escherichia coli when inducible synthetic constructs are expressed. We observed that native promoters related to the heat-shock response activated expression rapidly in response to synthetic expression, regardless of the construct. Using these promoters, we built a dCas9-based feedback-regulation system that automatically adjusts the expression of a synthetic construct in response to burden. Cells equipped with this general-use controller maintained their capacity for native gene expression to ensure robust growth and thus outperformed unregulated cells in terms of protein yield in batch production. This engineered feedback is to our knowledge the first example of a universal, burden-based biomolecular control system and is modular, tunable and portable.

Quantifying cellular capacity identifies gene expression designs with reduced burden.

Heterologous gene expression can be a significant burden for cells. Here we describe an in vivo monitor that tracks changes in the capacity of Escherichia coli in real time and can be used to assay the burden imposed by synthetic constructs and their parts. We identify construct designs with reduced burden that predictably outperformed less efficient designs, despite having equivalent output.

Simultaneous Plate-Reader Characterization of Promoter Activity and Cell Growth in Engineered Mammalian Cells.

Automated high-throughput methods that support tracking of mammalian cell growth are currently needed to advance cell line characterization and identification of desired genetic components required for cell engineering. Here, we describe a high-throughput noninvasive assay based on plate reader measurements. The assay relies on the change in absorbance of the pH indicator phenol red. We show that its basic and acidic absorbance profiles can be converted into a cell growth index consistent with cell count profiles, and that, by adopting a computational pipeline and calibration measurements, it is possible to identify a conversion that enables prediction of cell numbers from plate measurements alone. The assay is suitable for growth characterization of both suspension and adherent cell lines when these are grown under different environmental conditions and treated with chemotherapeutic drugs. The method also supports characterization of stably engineered cell lines and identification of desired promoters based on fluorescence output.

Engineered Bacteria as Living Biosensors in Dermal Tattoos.

Dermal tattoo biosensors are promising platforms for real-time monitoring of biomarkers, with skin used as a diagnostic interface. Traditional tattoo sensors have utilized small molecules as biosensing elements. However, the rise of synthetic biology has enabled the potential employment of engineered bacteria as living analytical tools. Exploiting engineered bacterial sensors will allow for potentially more sensitive detection across a broad biomarker range, with advanced processing and sense/response functionalities using genetic circuits. Here, the interfacing of bacterial biosensors as living analytics in tattoos is shown. Engineered bacteria are encapsulated into micron-scale hydrogel beads prepared through scalable microfluidics. These biosensors can sense both biochemical cues (model biomarkers) and biophysical cues (temperature changes, using RNA thermometers), with fluorescent readouts. By tattooing beads into skin models and confirming sensor activity post-tattooing, our study establishes a foundation for integrating bacteria as living biosensing entities in tattoos.

High-Throughput Spectroscopic Analysis of mRNA Capping Level.

Eukaryotic mRNAs are characterized by terminal 5' cap structures and 3' polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a novel biosensor method designed to detect the presence of both cap structures and polyadenylation sites on mRNA molecules. This novel biosensor is sensitive to mRNA degradation and can quantitatively determine capping levels of mRNA molecules within a mixture of capped and uncapped mRNA molecules. The biosensor displays a constant dynamic range between 254 nt and 6507 nt with reproducible sensitivity to increases in capping level of at least 20% and a limit of detection of 2.4 pmol of mRNA. Overall, the biosensor can provide key information about mRNA quality before mammalian cell transfection.

Mammalian cell growth characterisation by a non-invasive plate reader assay.

Automated and non-invasive mammalian cell analysis is currently lagging behind due to a lack of methods suitable for a variety of cell lines and applications. Here, we report the development of a high throughput non-invasive method for tracking mammalian cell growth and performance based on plate reader measurements. We show the method to be suitable for both suspension and adhesion cell lines, and we demonstrate it can be adopted when cells are grown under different environmental conditions. We establish that the method is suitable to inform on effective drug treatments to be used depending on the cell line considered, and that it can support characterisation of engineered mammalian cells over time. This work provides the scientific community with an innovative approach to mammalian cell screening, also contributing to the current efforts towards high throughput and automated mammalian cell engineering.

Resource-aware construct design in mammalian cells.

Resource competition can be the cause of unintended coupling between co-expressed genetic constructs. Here we report the quantification of the resource load imposed by different mammalian genetic components and identify construct designs with increased performance and reduced resource footprint. We use these to generate improved synthetic circuits and optimise the co-expression of transfected cassettes, shedding light on how this can be useful for bioproduction and biotherapeutic applications. This work provides the scientific community with a framework to consider resource demand when designing mammalian constructs to achieve robust and optimised gene expression.

Engineering Sensors for Gene Expression Burden.

RNA-seq enables the analysis of gene expression profiles across different conditions and organisms. Gene expression burden slows down growth, which results in poor predictability of gene constructs and product yields. Here, we describe how we applied RNA-seq to study the transcriptional profiles of Escherichia coli when burden is elicited during heterologous gene expression. We then present how we selected early responsive promoters from our RNA-seq results to design sensors for gene expression burden. Finally, we describe how we used one of these sensors to develop a burden-driven feedback regulator to improve cellular fitness in engineered E. coli.

Finite Element Modeling of Bond Behavior of FRP and Steel Plates.

Strengthening systems for existing reinforced concrete (RC) structures are increasingly needed due to several problems such as degradation of materials over the time, underdesign, serviceability or seismic upgrading, or new code requirements. In the last decades, strengthening by fibers composite materials applied with various techniques (FRP, FRCM, NSM) were largely investigated and theoretical formulations have been introduced in national and international design guidelines. Although they are an excellent strengthening solution, steel plates may represent still a valid traditional alternative, due to low costs, ductile stress-strain behavior, simple and fast mounting with possibility of reusing the material. Guidelines for a correct design are still lack and, therefore, detailed models and design formulas are needed. In this paper, the bond behavior at the plate-concrete interface, which plays a key role for the effectiveness of the strengthening system, is analyzed by means of 3D finite element models calibrated on experimental results available in literature. Parametric analyses were carried out by changing some meaningful parameters.

Literature Review of the In-Plane Behavior of Masonry Walls: Theoretical vs. Experimental Results.

In-plane strength of masonry walls is affected by the resistant mechanisms activated in the walls, i.e., related to flexural or shear behavior. The latter one can occur in the walls according to different failure modes depending on both mortar and unit strengths and on the type of assembling, i.e., 'regular' or 'irregular' texture. In this paper, a critical review of the existing design formulations for the in-plane strength of masonry walls is firstly presented, with important information on the achievable failure modes depending on the geometrical and mechanical features of the masonry fabric. Then, experimental tests are collected from the literature and a comparison between theoretical and experimental results is carried out. The presented analyses are aimed to highlight the differences between the existing formulations and to identify the most suitable ones.

In-Plane Behaviour of Masonry Walls: Numerical Analysis and Design Formulations.

This paper presents the results of several numerical analyses aimed at investigating the in-plane resistance of masonry walls by means of two modelling approaches: a finite element model (FEM) and a discrete macro-element model (DMEM). Non-linear analyses are developed, in both cases, by changing the mechanical properties of masonry (compressive and tensile strengths, fracture energy in compression and tension, shear strength) and the value of the vertical compression stress applied on the walls. The reliability of both numerical models is firstly checked by means of comparisons with experimental tests available in the literature. The analyses show that the numerical results provided by the two modelling approaches are in good agreement, in terms of both failure loads and modes, while some differences are observed in their load-displacement curves, especially in the non-linear field. Finally, the numerical in-plane resistances are compared with the theoretical formulations provided by the Italian building code for both flexural and shear failure modes and an amendment for the shape factor 'b' introduced in the code formulation for squat walls is proposed.

Genetic Toolkits to Design and Build Mammalian Synthetic Systems.

Construction of DNA-encoded programs is central to synthetic biology and the chosen method often determines the time required to design and build constructs for testing. Here, we describe and summarise key features of the available toolkits for DNA construction for mammalian cells. We compare the different cloning strategies based on their complexity and the time needed to generate constructs of different sizes, and we reflect on why Golden Gate toolkits now dominate due to their modular design. We look forward to future advances, including accessory packs for cloning toolkits that can facilitate editing, orthogonality, advanced regulation, and integration into synthetic chromosome construction.

Non-Histone Protein Methylation: Biological Significance and Bioengineering Potential.

Protein methylation is a key post-translational modification whose effects on gene expression have been intensively studied over the last two decades. Recently, renewed interest in non-histone protein methylation has gained momentum for its role in regulating important cellular processes and the activity of many proteins, including transcription factors, enzymes, and structural complexes. The extensive and dynamic role that protein methylation plays within the cell also highlights its potential for bioengineering applications. Indeed, while synthetic histone protein methylation has been extensively used to engineer gene expression, engineering of non-histone protein methylation has not been fully explored yet. Here, we report the latest findings, highlighting how non-histone protein methylation is fundamental for certain cellular functions and is implicated in disease, and review recent efforts in the engineering of protein methylation.

Initial cell density encodes proliferative potential in cancer cell populations.

Individual cells exhibit specific proliferative responses to changes in microenvironmental conditions. Whether such potential is constrained by the cell density throughout the growth process is however unclear. Here, we identify a theoretical framework that captures how the information encoded in the initial density of cancer cell populations impacts their growth profile. By following the growth of hundreds of populations of cancer cells, we found that the time they need to adapt to the environment decreases as the initial cell density increases. Moreover, the population growth rate shows a maximum at intermediate initial densities. With the support of a mathematical model, we show that the observed interdependence of adaptation time and growth rate is significantly at odds both with standard logistic growth models and with the Monod-like function that governs the dependence of the growth rate on nutrient levels. Our results (i) uncover and quantify a previously unnoticed heterogeneity in the growth dynamics of cancer cell populations; (ii) unveil how population growth may be affected by single-cell adaptation times; (iii) contribute to our understanding of the clinically-observed dependence of the primary and metastatic tumor take rates on the initial density of implanted cancer cells.

Growth Defects and Loss-of-Function in Synthetic Gene Circuits.

Synthetic gene circuits perturb the physiology of their cellular host. The extra load on endogenous processes shifts the equilibrium of resource allocation in the host, leading to slow growth and reduced biosynthesis. Here we built integrated host-circuit models to quantify growth defects caused by synthetic gene circuits. Simulations reveal a complex relation between circuit output and cellular capacity for gene expression. For weak induction of heterologous genes, protein output can be increased at the expense of growth defects. Yet for stronger induction, cellular capacity reaches a tipping point, beyond which both gene expression and growth rate drop sharply. Extensive simulations across various growth conditions and large regions of the design space suggest that the critical capacity is a result of ribosomal scarcity. We studied the impact of growth defects on various gene circuits and transcriptional logic gates, which highlights the extent to which cellular burden can limit, shape, and even break down circuit function. Our approach offers a comprehensive framework to assess the impact of host-circuit interactions in silico, with wide-ranging implications for the design and optimization of bacterial gene circuits.

Overloaded and stressed: whole-cell considerations for bacterial synthetic biology.

The predictability and robustness of engineered bacteria depend on the many interactions between synthetic constructs and their host cells. Expression from synthetic constructs is an unnatural load for the host that typically reduces growth, triggers stresses and leads to decrease in performance or failure of engineered cells. Work in systems and synthetic biology has now begun to address this through new tools, methods and strategies that characterise and exploit host-construct interactions in bacteria. Focusing on work in E. coli, we review here a selection of the recent developments in this area, highlighting the emerging issues and describing the new solutions that are now making the synthetic biology community consider the cell just as much as they consider the construct.

Sensing the Right Time to Be Productive.

Engineered E. coli can be made to autonomously switch from growth to production by a modular two-gate system that reduces the burden of biosynthesis.