Publications
28 results found
Redwood-Sawyerr C, Aw R, Di Blasi R, et al., 2024, High-throughput spectroscopic analysis of mRNA capping level, Mammalian Synthetic Systems, Publisher: Humana New York, NY, Pages: 269-278, ISBN: 978-1-0716-3718-0
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.
Grob A, Enrico Bena C, Di Blasi R, et al., 2024, Mammalian cell growth characterisation by a non-invasive plate reader assay, Nature Communications, Vol: 15, ISSN: 2041-1723
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.
Di Blasi R, Pisani M, Tedeschi F, et al., 2023, Resource-aware construct design in mammalian cells, Nature Communications, Vol: 14, ISSN: 2041-1723
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.
Monck C, Elani Y, Ceroni F, 2022, Cell-free protein synthesis: biomedical applications and future perspectives, Chemical Engineering Research and Design, Vol: 177, Pages: 653-658, ISSN: 0263-8762
The use of cell-free protein synthesis (CFPS) has become increasingly widespread in synthetic biology over recent years, providing an effective platform for the study and engineering of cellular processes. The versatility and portability of CFPS systems have also boosted their potential for usage outside of the laboratory in a wide number of applications, from construct prototyping to bioproduction. CFPS is particularly well suited to biomedical applications, such as the production of clinical molecules and vaccines. It can also be integrated with additional technologies such as microfluidics and liposomal encapsulation to provide a new route for on-demand therapeutic expression. In this review we outline the key features of CFPS that make it a powerful platform for biomedical applications. We also discuss existing limitations with respect to the use of CFPS in the production of complex protein products and the limited production capacity of current systems. Addressing these will be integral in expanding the application of CFPS in biotherapy
Grob A, Di Blasi R, Ceroni F, 2021, Experimental tools to reduce the burden of bacterial synthetic biology, Current Opinion in Systems Biology, Vol: 28, ISSN: 2452-3100
Cellular burden limits the applications of bacterial synthetic biology. Experimental approaches for burden minimisation have recently become available. Tools to identify construct design with low footprint on the host include capacity monitors that quantify cellular capacity, high-throughput approaches and cell-free systems for construct prototyping. Orthogonal ribosomes and feedback controllers are instead useful to seek control of resource allocation and lower burden. Other approaches include genome reduction to increase the available resource pool and synthetic addiction to couple cell fitness and product accumulation. However, controlling the cellular response to exogenous expression is still a challenge, and more tools are needed in order to widen the applications of synthetic biology. Further effort that combines novel evolutionary data with burden-aware tools can set the foundation to increase the stability and robustness of future genetic systems.
Di Blasi R, Zouein A, Ellis T, et al., 2021, Genetic toolkits to design and build mammalian synthetic systems, Trends in Biotechnology, Vol: 39, Pages: 1004-1018, ISSN: 0167-7799
Construction of DNA-encoded programs is central to synthetic biology and the chosen method oftendetermines the time required to design and build constructs for testing. Here we describe and summarisekey features of the available toolkits for DNA construction for mammalian cells. We compare the differentcloning 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 tofuture advances, including accessory packs to cloning toolkits that can facilitating editing, orthogonality,advanced regulation, and integration into synthetic chromosome construction.
Di Blasi R, Marbiah M, Siciliano V, et al., 2021, A call for caution in analysing mammalian co-transfection experiments and implications of resource competition in data misinterpretation, Nature Communications, Vol: 12, ISSN: 2041-1723
Transient transfections are routinely used in basic and synthetic biology studies to unravel pathway regulation and to probe and characterise circuit designs. As each experiment has a component of intrinsic variability, reporter gene expression is usually normalized with co-delivered genes that act as transfection controls. Recent reports in mammalian cells highlight how resource competition for gene expression leads to biases in data interpretation, with a direct impact on co-transfection experiments. Here we define the connection between resource competition and transient transfection experiments and discuss possible alternatives. Our aim is to raise awareness within the community and stimulate discussion to include such considerations in future experimental designs, for the development of better transfection controls.
Bena CE, Del Giudice M, Grob A, et al., 2021, Initial cell density encodes proliferative potential in cancer cell populations, Scientific Reports, Vol: 11, Pages: 1-11, ISSN: 2045-2322
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.
Di Blasi R, Blyuss O, Timms JF, et al., 2021, Non-histone protein methylation: biological significance and bioengineering potential, ACS Chemical Biology, Vol: 16, Pages: 238-250, ISSN: 1554-8929
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.
Boo A, Ceroni F, 2021, Engineering sensors for gene expression burden, Methods in Molecular Biology, Publisher: Humana Press, Pages: 313-330
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.
Evans SW, Beal J, Berger K, et al., 2020, Embrace experimentation in biosecurity governance, Science, Vol: 368, Pages: 138-140, ISSN: 0036-8075
Ledesma-Amaro R, Nikel PI, Ceroni F, 2020, Editorial: synthetic biology-guided metabolic engineering, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
Nikolados E, Weisse A, Ceroni F, et al., 2019, Growth defects and loss-of-function in synthetic gene circuits, ACS Synthetic Biology, Vol: 8, Pages: 1231-1240, ISSN: 2161-5063
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.
Ceroni F, Ellis T, 2018, The challenges facing synthetic biology in eukaryotes, Nature Reviews Molecular Cell Biology, Vol: 19, Pages: 481-482, ISSN: 1471-0072
Synthetic biology is maturing into a true engineering discipline for model microorganisms, but remains far from straightforward for most eukaryotes. Here, we outline the key challenges facing those trying to engineer biology across eukaryota and suggest areas of focus that will aid future progress.
Enrico Bena C, Grob A, Isalan M, et al., 2018, Commentary: Synthetic Addiction Extends the Productive Life Time of Engineered Escherichia coli Populations, Frontiers in Bioengineering and Biotechnology, Vol: 6, ISSN: 2296-4185
A commentary on Synthetic addiction extends the productive life time of engineered Escherichia coli populations by Rugbjerg, P., Sarup-Lytzen, K., Nagy, M., and Sommer, M. O. A. (2018). Proc. Natl. Acad. Sci. U.S.A. 115, 2347–2352. doi: 10.1073/pnas.1718622115Bioproduction is the process of producing added-value chemicals on large-scale using cells as biological factories. Cellular burden represents a significant problem in the scaling of fermentation processes from proof-of-concept to long-term cultures, as the load of heterologous gene expression and depletion of the cell intracellular resources cause unpredictable cellular physiological changes that can lead to decreased growth and lower production yields (Borkowski et al., 2016; Liu et al., 2018). One possible cause of the observed decreased bioproduct recovery in many bioprocessing applications is the accumulation of mutations in the employed genetic program. These mutations often lead to loss of production and rise of non-producing populations that grow better and easily overtake the growth of producing cells (Rugbjerg et al., 2018b).In a recent paper in PNAS, Rugbjerg et al. (2018b) developed a strategy to limit the enrichment of non-producing cell populations in bioproduction-employed cell cultures by placing the genes for key growth intermediates under the control of a promoter responsive to the bioproduct being made. This strategy known as product addiction was tested in E. coli engineered to produce mevalonic acid in long-term cultivations (Figure 1).
Ceroni F, Boo A, Furini S, et al., 2018, Burden-driven feedback control of gene expression, Nature Methods, Vol: 15, Pages: 387-393, ISSN: 1548-7091
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.
Ceroni F, Furini S, Gorochowski T, et al., 2017, Burden-driven feedback control of gene expression, Publisher: Cold Spring Harbor Laboratory
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.
Ceroni F, Blount BA, Ellis T, 2016, Sensing the Right Time to Be Productive, Cell Systems, Vol: 3, Pages: 116-117, ISSN: 2405-4720
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.
Borkowski O, Ceroni F, Stan GB, et al., 2016, Overloaded and stressed: whole-cell considerations for bacterial synthetic biology., Current Opinion in Microbiology, Vol: 33, Pages: 123-130, ISSN: 1879-0364
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.
Ceroni F, Carbonell P, François JM, et al., 2015, Editorial - Synthetic Biology: Engineering Complexity and Refactoring Cell Capabilities., Frontiers in Bioengineering and Biotechnology, Vol: 3, ISSN: 2296-4185
Ceroni F, Algar R, Stan G-B, et al., 2015, Quantifying cellular capacity identifies gene expression designs with reduced burden, Nature Methods, Vol: 12, Pages: 415-418, ISSN: 1548-7105
Heterologous gene expression can be a significant burden forcells. Here we describe an in vivo monitor that tracks changesin the capacity of Escherichia coli in real time and can be usedto assay the burden imposed by synthetic constructs and theirparts. We identify construct designs with reduced burden thatpredictably outperformed less efficient designs, despite havingequivalent output.
Pothoulakis G, Ceroni F, Reeve B, et al., 2014, The spinach RNA aptamer as a characterization tool for synthetic biology, ACS Synthetic Biology, Vol: 3, Pages: 182-187, ISSN: 2161-5063
Characterization of genetic control elements is essential for the predictable engineering of synthetic biology systems. The current standard for in vivo characterization of control elements is through the use of fluorescent reporter proteins such as green fluorescent protein (GFP). Gene expression, however, involves not only protein production but also the production of mRNA. Here, we present the use of the Spinach aptamer sequence, an RNA mimic of GFP, as a tool to characterize mRNA expression in Escherichia coli. We show how the aptamer can be incorporated into gene expression cassettes and how co-expressing it with a red fluorescent protein (mRFP1) allows, for the first time, simultaneous measurement of mRNA and protein levels from engineered constructs. Using flow cytometry, we apply this tool here to evaluate ribosome binding site sequences and promoters and use it to highlight the differences in the temporal behavior of transcription and translation.
Selvaggio G, Ceroni F, Giordano E, et al., 2013, Evaluation of the Expression Level of a Fluorescent Protein in Single Cells through Digital Image Processing, 5th Latin American Congress on Biomedical Engineering (CLAIB 2011), Publisher: SPRINGER, Pages: 1007-+, ISSN: 1680-0737
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Ceroni F, Furini S, Stefan A, et al., 2012, A Synthetic Post-transcriptional Controller To Explore the Modular Design of Gene Circuits, ACS Synthetic Biology, Vol: 1, Pages: 163-171, ISSN: 2161-5063
The assembly from modular parts is an efficient approach for creating new devices in Synthetic Biology. In the "bottom-up" designing strategy, modular parts are characterized in advance, and then mathematical modeling is used to predict the outcome of the final device. A prerequisite for bottom-up design is that the biological parts behave in a modular way when assembled together. We designed a new synthetic device for post-transcriptional regulation of gene expression and tested if the outcome of the device could be described from the features of its components. Modular parts showed unpredictable behavior when assembled in different complex circuits. This prevented a modular description of the device that was possible only under specific conditions. Our findings shed doubts into the feasibility of a pure bottom-up approach in synthetic biology, highlighting the urgency for new strategies for the rational design of synthetic devices.
Haynes KA, Ceroni F, Flicker D, et al., 2012, A sensitive switch for visualizing natural gene silencing in single cells, ACS Synthetic Biology, Vol: 1, Pages: 99-106, ISSN: 2161-5063
RNA interference is a natural gene expression silencing system that appears throughout the tree of life. As the list of cellular processes linked to RNAi grows, so does the demand for tools to accurately measure RNAi dynamics in living cells. We engineered a synthetic RNAi sensor that converts this negative regulatory signal into a positive output in living mammalian cells, thereby allowing increased sensitivity and activation. Furthermore, the circuit’s modular design allows potentially any microRNA of interest to be detected. We demonstrated that the circuit responds to an artificial microRNA and becomes activated when the RNAi target is replaced by a natural microRNA target (miR-34) in U2OS osteosarcoma cells. Our studies extend the application of rationally designed synthetic switches to RNAi, providing a sensitive way to visualize the dynamics of RNAi activity rather than just the presence of miRNA molecules.
Ceroni F, Furini S, Passini E, et al., 2011, A synthetic molecular tool for post-transcriptional control of gene expression in E. coli., Annual Meeting of the American-Society-for-Cell-Biology (ASCB), Publisher: AMER SOC CELL BIOLOGY, ISSN: 1059-1524
Ceroni F, Furini S, Giordano E, et al., 2010, Rational design of modular circuits for gene transcription: a test of the bottom-up approach, Journal of Biological Engineering, Vol: 4, ISSN: 1754-1611
BACKGROUND: Most of synthetic circuits developed so far have been designed by an ad hoc approach, using a small number of components (i.e. LacI, TetR) and a trial and error strategy. We are at the point where an increasing number of modular, inter-changeable and well-characterized components is needed to expand the construction of synthetic devices and to allow a rational approach to the design. RESULTS: We used interchangeable modular biological parts to create a set of novel synthetic devices for controlling gene transcription, and we developed a mathematical model of the modular circuits. Model parameters were identified by experimental measurements from a subset of modular combinations. The model revealed an unexpected feature of the lactose repressor system, i.e. a residual binding affinity for the operator site by induced lactose repressor molecules. Once this residual affinity was taken into account, the model properly reproduced the experimental data from the training set. The parameters identified in the training set allowed the prediction of the behavior of networks not included in the identification procedure. CONCLUSIONS: This study provides new quantitative evidences that the use of independent and well-characterized biological parts and mathematical modeling, what is called a bottom-up approach to the construction of gene networks, can allow the design of new and different devices re-using the same modular parts.
Ceroni F, Furini S, Cavalcanti S, 2009, A computational model of gene expression in an inducible synthetic circuit, Biocomputing 2010, Publisher: World Scientific Publishing Company, Pages: 409-420, ISSN: 2335-6936
Synthetic biology aims to the rational design of gene circuits with predictable behaviours. Great efforts have been done so far to introduce in the field mathematical models that could facilitate the design of synthetic networks. Here we present a mathematical model of a synthetic gene-circuit with a negative feedback. The closed loop configuration allows the control of transcription by an inducer molecule (IPTG). Escherichia coli bacterial cells were transformed and expression of a fluorescent reporter (GFP) was measured for different inducer levels. Computer model simulations well reproduced the experimental induction data, using a single fitting parameter. Independent genetic components were used to assemble the synthetic circuit. The mathematical model here presented could be useful to predict how changes in these genetic components affect the behaviour of the synthetic circuit.
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