82 results found
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, ISSN: 2041-1723
Aw R, De Wachter C, Laukens B, et al., 2021, Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris, Traffic, Vol: 22, Pages: 48-63, ISSN: 1398-9219
The structural organization of the Golgi stacks in mammalian cells is intrinsically linked to function, including glycosylation, but the role of morphology is less clear in lower eukaryotes. Here we investigated the link between the structural organization of the Golgi and secretory pathway function using Pichia pastoris as a model system. To unstack the Golgi cisternae, we disrupted 18 genes encoding proteins in the secretory pathway without loss of viability. Using biosensors, confocal microscopy and transmission electron microscopy we identified three strains with irreversible perturbations in the stacking of the Golgi cisternae, all of which had disruption in genes that encode proteins with annotated function as or homology to calcium/calcium permeable ion channels. Despite this, no variation in the secretory pathway for ER size, whole cell glycomics or recombinant protein glycans was observed. Our investigations showed the robust nature of the secretory pathway in P. pastoris and suggest that Ca2+ concentration, homeostasis or signalling may play a significant role for Golgi stacking in this organism and should be investigated in other organisms.
Heide C, Buldum G, Moya-Ramirez I, et al., 2021, Design, development and optimisation of a functional mammalian cell-free protein synthesis platform, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
In this paper, we describe the stepwise development of a cell-free protein synthesis (CFPS) platform derived from cultured Chinese hamster ovary (CHO) cells. We provide a retrospective summary of the design challenges we faced, and the optimized methods developed for the cultivation of cells and the preparation of translationally active lysates. To overcome low yields, we developed procedures to supplement two accessory proteins, GADD34 and K3L, into the reaction to prevent deactivation of the translational machinery by phosphorylation. We compared different strategies for implementing these accessory proteins including two variants of the GADD34 protein to understand the potential trade-offs between yield and ease of implementation. Addition of the accessory proteins increased yield of turbo Green Fluorescent Protein (tGFP) by up to 100-fold depending on which workflow was used. Using our optimized protocols as a guideline, users can successfully develop their own functional CHO CFPS system, allowing for broader application of mammalian CFPS.
Shmool TA, Martin LK, Clarke CJ, et al., 2021, Exploring conformational preferences of proteins: ionic liquid effects on the energy landscape of avidin, CHEMICAL SCIENCE, Vol: 12, Pages: 196-209, ISSN: 2041-6520
Keck FD, Polizzi K, 2021, Microbial interventions are an easier alternative to engineer higher organisms, Microbial Biotechnology, Vol: 14, Pages: 26-30, ISSN: 1751-7907
Advances in synthetic biology have made microbes easier to engineer than ever before. However, synthetic biology in animals and plants has lagged behind. Since it is now known that the phenotype of higher organisms depends largely on their microbiota, we propose that this is an easier route to achieving synthetic biology applications in these organisms.
Moya-Ramirez I, Bouton C, Kontoravdi C, et al., 2020, High resolution biosensor to test the capping level and integrity of mRNAs, Nucleic Acids Research, Vol: 48, Pages: 1-11, ISSN: 0305-1048
5 Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing,translation initiation and stability. Here we describea biosensor designed to detect the presence of capstructures on mRNAs that is also sensitive to mRNAdegradation, so uncapped or degraded mRNAs canbe detected in a single step. The biosensor is basedon a chimeric protein that combines the recognitionand transduction roles in a single molecule. The mainfeature of this sensor is its simplicity, enabling semiquantitative analyses of capping levels with minimalinstrumentation. The biosensor was demonstratedto detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic rangeremained constant with RNAs ranging in size from250 nt to approximately 2700 nt and the biosensorwas able to detect variations in the capping level inincrements of at least 20%, with a limit of detection of2.4 pmol. Remarkably, it also can be applied to morecomplex analytes, such mRNA vaccines and mRNAstranscribed in vivo. This biosensor is an innovativeexample of a technology able to detect analyticallychallenging structures such as mRNA caps. It couldfind application in a variety of scenarios, from qualityanalysis of mRNA-based products such as vaccinesto optimization of in vitro capping reactions.
Aw R, Spice AJ, Polizzi K, 2020, Methods for expression of recombinant proteins using a Pichia pastoris cell-free system, Current protocols in protein science, Vol: 102, ISSN: 1934-3655
Cell‐free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell‐free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell‐free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post‐translational modifications. Here, we describe methods for conducting cell‐free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription‐translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike.
Spice AJ, Aw R, Bracewell DG, et al., 2020, Improving the reaction mix of a Pichia pastoris cell-free system using a design of experiments approach to minimise experimental effort, Synthetic and Systems Biotechnology, Vol: 5, Pages: 137-144, ISSN: 2405-805X
A renaissance in cell-free protein synthesis (CFPS) is underway, enabled by the acceleration and adoption of synthetic biology methods. CFPS has emerged as a powerful platform technology for synthetic gene network design, biosensing and on-demand biomanufacturing. Whilst primarily of bacterial origin, cell-free extracts derived from a variety of host organisms have been explored, aiming to capitalise on cellular diversity and the advantageous properties associated with those organisms. However, cell-free extracts produced from eukaryotes are often overlooked due to their relatively low yields, despite the potential for improved protein folding and posttranslational modifications. Here we describe further development of a Pichia pastoris cell-free platform, a widely used expression host in both academia and the biopharmaceutical industry. Using a minimised Design of Experiments (DOE) approach, we were able to increase the productivity of the system by improving the composition of the complex reaction mixture. This was achieved in a minimal number of experimental runs, within the constraints of the design and without the need for liquid-handling robots. In doing so, we were able to estimate the main effects impacting productivity in the system and increased the protein synthesis of firefly luciferase and the biopharmaceutical HSA by 4.8-fold and 3.5-fold, respectively. This study highlights the P. pastoris-based cell-free system as a highly productive eukaryotic platform and displays the value of minimised DOE designs.
Gschwend FJ, Hennequin LM, Brandt-Talbot A, et al., 2020, Towards an environmentally and economically sustainable biorefinery: heavy metal contaminated waste wood as a low-cost feedstock in a low-cost ionic liquid process, Green Chemistry, Vol: 22, Pages: 5032-5041, ISSN: 1463-9262
In the present study, we used a low-cost protic ionic liquid, 1-methylimidazolium chloride, to simultaneously fractionate heavy metal contaminated wood and extract the metals from the wood at elevated temperature and short reaction time. This treatment selectively dissolves the lignin and hemicellulose in the biomass, leaving a solid cellulose-rich pulp, while coordinating and extracting 80–100% of the metal species present in the wood in a one-pot process. The lignin stream was recovered from the liquor and the cellulose was hydrolysed and then fermented into ethanol. The ionic liquid was recycled 6 times and the metals were recovered from the liquor via electrodeposition. This is the first time that highly contaminated waste wood has been integrated into a process which does not produce a contaminated waste stream, but instead valorises the wood as a feedstock for renewable chemicals, materials and fuels, while efficiently recovering the metals, converting a toxic environmental hazard into a rich source of biorenewables. We have therefore used an otherwise problematic waste as a low-cost lignocellulsoic feedstock for a circular bioeconomy concept.
Arpino JAJ, Polizzi KM, 2020, A modular method for directing protein self-assembly, ACS Synthetic Biology, Vol: 9, Pages: 993-1002, ISSN: 2161-5063
Proteins are versatile macromolecules with diverse structure, charge, and function. They are ideal building blocks for biomaterials for drug delivery, biosensing, or tissue engineering applications. Simultaneously, the need to develop green alternatives to chemical processes has led to renewed interest in multienzyme biocatalytic routes to fine, specialty, and commodity chemicals. Therefore, a method to reliably assemble protein complexes using protein-protein interactions would facilitate the rapid production of new materials. Here we show a method for modular assembly of protein materials using a supercharged protein as a scaffolding "hub" onto which target proteins bearing oppositely charged domains have been self-assembled. The physical properties of the material can be tuned through blending and heating and disassembly triggered using changes in pH or salt concentration. The system can be extended to the synthesis of living materials. Our modular method can be used to reliably direct the self-assembly of proteins using small charged tag domains that can be easily encoded in a fusion protein.
Bui-Le L, Clarke CJ, Bröhl A, et al., 2020, Revealing the complexity of ionic liquid-protein interactions through a multi-technique investigation, Communications Chemistry, Vol: 3, ISSN: 2399-3669
Ionic liquids offer exciting possibilities for biocatalysis as solvent properties provide rare opportunities for customizable, energy-efficient bioprocessing. Unfortunately, proteins and enzymes are generally unstable in ionic liquids and several attempts have been made to explain why; however, a comprehensive understanding of the ionic liquid–protein interactions remains elusive. Here, we present an analytical framework (circular dichroism (CD), fluorescence, ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR) spectroscopies, and small-angle X-ray scattering (SAXS)) to probe the interactions, structure, and stability of a model protein (green fluorescent protein (GFP)) in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts. We demonstrate that measuring protein stability requires a similar holistic analytical framework, as opposed to single-technique assessments that provide misleading conclusions. We reveal information on site-specific ionic liquid–protein interactions, revealing that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding. Robust frameworks such as this are critical to advancing non-aqueous biocatalysis and avoiding pitfalls associated with single-technique investigations.
Moore SJ, Lai H-E, Kelwick RJR, et al., 2020, Correction to EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology., ACS Synthetic Biology, ISSN: 2161-5063
It has been brought to our attention that the original article contains a typographical error within Figure 1B, part ii. One of the 4-bp overhangs reads “GGAC” and should instead be “GTAC”, as is consistent throughout the original manuscript and deposited AddGene sequences.
Riangrungroj P, Polizzi KM, 2020, BeQuIK (Biosensor Engineered Quorum Induced Killing): designer bacteria for destroying recalcitrant biofilms., Microbial Biotechnology, Vol: 13, Pages: 311-314, ISSN: 1751-7915
This opinion piece describes a new design for the remediation of recalcitrant biofilms. It builds on previous work to develop engineered E. coli that recognize quorum sensing signals from pathogens in a biofilm and secrete toxins in response. To solve the challenge of dilute signalling molecules, we propose to use nanobodies and enzymes displayed on the surface of the cells to localize them to the biofilm and degrade the extracellular polymeric substances, thus creating a solution with better 'seek and destroy' capabilities.
Spice AJ, Aw R, Bracewell DG, et al., 2020, Synthesis and assembly of Hepatitis B virus-like particles in a Pichia pastoris cell-free system, Frontiers in Bioengineering and Biotechnology, Vol: 8, ISSN: 2296-4185
Virus-like particles (VLPs) are supramolecular protein assemblies with the potential for unique and exciting applications in synthetic biology and medicine. Despite the attention VLPs have gained thus far, considerable limitations still persist in their production. Poorly scalable manufacturing technologies and inconsistent product architectures continue to restrict the full potential of VLPs. Cell-free protein synthesis (CFPS) offers an alternative approach to VLP production and has already proven to be successful, albeit using extracts from a limited number of organisms. Using a recently developed Pichia pastoris-based CFPS system, we have demonstrated the production of the model Hepatitis B core antigen VLP as a proof-of-concept. The VLPs produced in the CFPS system were found to have comparable characteristics to those previously produced in vivo and in vitro. Additionally, we have developed a facile and rapid synthesis, assembly and purification methodology that could be applied as a rapid prototyping platform for vaccine development or synthetic biology applications. Overall the CFPS methodology allows far greater throughput, which will expedite the screening of optimal assembly conditions for more robust and stable VLPs. This approach could therefore support the characterization of larger sample sets to improve vaccine development efficiency.
Riangrungroj P, Bever CS, Hammock BD, et al., 2019, A label-free optical whole-cell Escherichia coli biosensor for the detection of pyrethroid insecticide exposure, Scientific Reports, Vol: 9, Pages: 1-9, ISSN: 2045-2322
There is a growing need for low-cost, portable technologies for the detection of threats to the environment and human health. Here we propose a label-free, optical whole-cell Escherichia coli biosensor for the detection of 3-phenoxybenzoic acid (3-PBA), a biomarker for monitoring human exposure to synthetic pyrethroid insecticides. The biosensor functions like a competitive ELISA but uses whole-cells surface displaying an anti-3-PBA VHH as the detection element. When the engineered cells are mixed with 3-PBA-protein conjugate crosslinking that can be visually detected occurs. Free 3-PBA in samples competes with these crosslinks, leading to a detectable change in the output. The assay performance was improved by coloring the cells via expression of the purple-blue amilCP chromoprotein and the VHH expression level was reduced to obtain a limit of detection of 3 ng/mL. The optimized biosensor exhibited robust function in complex sample backgrounds such as synthetic urine and plasma. Furthermore, lyophilization enabled storage of biosensor cells for at least 90 days without loss of functionality. Our whole-cell biosensor is simple and low-cost and therefore has potential to be further developed as a screening tool for monitoring exposure to pyrethroids in low-resource environments.
Marques MPC, Boyd AS, Polizzi K, et al., 2019, Microfluidic devices towards personalized health and wellbeing, JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, Vol: 94, Pages: 2412-2415, ISSN: 0268-2575
Kotidis P, Jedrzejewski P, Sou SN, et al., 2019, Model-based optimization of antibody galactosylation in CHO cell culture, Biotechnology and Bioengineering, Vol: 116, Pages: 1612-1626, ISSN: 0006-3592
Exerting control over the glycan moieties of antibody therapeutics is highly desirable from a product safety and batch-to-batch consistency perspective. Strategies to improve antibody productivity may compromise quality, while interventions for improving glycoform distribution can adversely affect cell growth and productivity. Process design therefore needs to consider the trade-off between preserving cellular health and productivity while enhancing antibody quality. In this work, we present a modeling platform that quantifies the impact of glycosylation precursor feeding - specifically that of galactose and uridine - on cellular growth, metabolism as well as antibody productivity and glycoform distribution. The platform has been parameterized using an initial training data set yielding an accuracy of ±5% with respect to glycoform distribution. It was then used to design an optimized feeding strategy that enhances the final concentration of galactosylated antibody in the supernatant by over 90% compared with the control without compromising the integral of viable cell density or final antibody titer. This work supports the implementation of Quality by Design towards higher-performing bioprocesses.
Moya-Ramirez I, Kontoravdi K, Polizzi K, 2019, Low-cost and user-friendly biosensor to test the integrity of mRNA molecules suitable for field applications, Biosensors and Bioelectronics, Vol: 137, Pages: 199-206, ISSN: 0956-5663
The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5’ end of the RNA under study and β-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-βLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products.
Aw R, Polizzi KM, 2019, Biosensor‐assisted engineering of a high‐yield Pichia pastoris cell‐free protein synthesis platform, Biotechnology and Bioengineering, Vol: 116, Pages: 656-666, ISSN: 0006-3592
Cell‐free protein synthesis (CFPS) has recently undergone a resurgence partly due to the proliferation of synthetic biology. The variety of hosts used for cell‐free extract production has increased, which harnesses the diversity of cellular biosynthetic, protein folding, and posttranslational modification capabilities available. Here we describe a CFPS platform derived from Pichia pastoris, a popular recombinant protein expression host both in academia and the biopharmaceutical industry. A novel ribosome biosensor was developed to optimize the cell extract harvest time. Using this biosensor we identified a potential bottleneck in ribosome content. Therefore, we undertook strain engineering to overexpress global regulators of ribosome biogenesis to increase in vitro protein production. CFPS extracts from the strain overexpressing FHL1 had a 3‐fold increase in recombinant protein yield compared to those from the wild‐type X33 strain. Furthermore, our novel CFPS platform can produce complex therapeutic proteins, as exemplified by the production of human serum albumin to a final yield of 48.1 μg mL‐1. Therefore, this work not only adds to the growing number of CFPS systems from diverse organisms, but also provides a blueprint for rapidly engineering new strains with increased productivity in vitro that could be applied to other organisms.
Kylilis N, Riangrungroj P, Lai H-E, et al., 2019, Whole-cell biosensor with tuneable limit of detection enables low-cost agglutination assays for medical diagnostic applications, ACS Sensors, Vol: 4, Pages: 370-378, ISSN: 2379-3694
Whole-cell biosensors can form the basis of affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing, but to date, the detection of analytes such as proteins that cannot easily diffuse across the cell membrane has been challenging. Here we developed a novel biosensing platform based on cell agglutination using an E. coli whole-cell biosensor surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design can detect a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations using straight-forward design rules and a mathematical model. Finally, we re-engineer our whole-cell biosensor for the detection of a medically relevant biomarker by the display of two different nanbodies against human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.
Heide C, Buldum G, Ces P, et al., 2019, Boosting the activity of CHO-based cell-free protein synthesis factories for high-throughput in vitro production of functional antibodies
Trantidou T, Dekker L, Polizzi K, et al., 2018, Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors, Interface Focus, Vol: 8, ISSN: 2042-8901
The design of vesicle microsystems as artificial cells (bottom-up synthetic biology) has traditionally relied on the incorporation of molecular components to impart functionality. These cell mimics have reduced capabilities compared with their engineered biological counterparts (top-down synthetic biology), as they lack the powerful metabolic and regulatory pathways associated with living systems. There is increasing scope for using whole intact cellular components as functional modules within artificial cells, as a route to increase the capabilities of artificial cells. In this feasibility study, we design and embed genetically engineered microbes (Escherichia coli) in a vesicle-based cell mimic and use them as biosensing modules for real-time monitoring of lactate in the external environment. Using this conceptual framework, the functionality of other microbial devices can be conferred into vesicle microsystems in the future, bridging the gap between bottom-up and top-down synthetic biology.
Kylilis N, Riangrungroj P, Lai H-E, et al., 2018, A low-cost biological agglutination assay for medical diagnostic applications, Publisher: American Chemical Society
Affordable, easy-to-use diagnostic tests that can be readily deployed for point-of-care (POC) testing are key in addressing challenges in the diagnosis of medical conditions and for improving global health in general. Ideally, POC diagnostic tests should be highly selective for the biomarker, user-friendly, have a flexible design architecture and a low cost of production. Here we developed a novel agglutination assay based on whole E. coli cells surface-displaying nanobodies which bind selectively to a target protein analyte. As a proof-of-concept, we show the feasibility of this design as a new diagnostic platform by the detection of a model analyte at nanomolar concentrations. Moreover, we show that the design architecture is flexible by building assays optimized to detect a range of model analyte concentrations supported using straight-forward design rules and a mathematical model. Finally, we re-engineer E. coli cells for the detection of a medically relevant biomarker by the display of two different antibodies against the human fibrinogen and demonstrate a detection limit as low as 10 pM in diluted human plasma. Overall, we demonstrate that our agglutination technology fulfills the requirement of POC testing by combining low-cost nanobody production, customizable detection range and low detection limits. This technology has the potential to produce affordable diagnostics for both field-testing in the developing world, emergency or disaster relief sites as well as routine medical testing and personalized medicine.
Aw R, McKay P, Shattock R, et al., 2018, A systematic analysis of the expression of the anti-HIV VRC01 antibody in Pichia pastoris through signal peptide optimization, Protein Expression and Purification, Vol: 149, Pages: 43-50, ISSN: 1046-5928
Pichia pastoris (Komagataella phaffi) has been used for recombinant protein production for over 30 years with over 5000 proteins reported to date. However, yields of antibody are generally low. We have evaluated the effect of secretion signal peptides on the production of a broadly neutralizing antibody (VRC01) to increase yield. Eleven different signal peptides, including the murine IgG1 signal peptide, were combinatorially evaluated for their effect on antibody titer. Strains using different combinations of signal peptides were identified that secreted approximately 2-7 fold higher levels of VRC01 than the previous best secretor, with the highest yield of 6.50 mg L-1 in shake flask expression. Interestingly it was determined that the highest yields were achieved when the murine IgG1 signal peptide was fused to the light chain, with several different signal peptides leading to high yield when fused to the heavy chain. Finally, we have evaluated the effect of using a 2A signal peptide to create a bicistronic vector in the attempt to reduce burden and increase transformation efficiency, but found it to give reduced yields compared to using two independent vectors.
Kylilis N, Tuza ZA, Stan G, et al., 2018, Tools for engineering coordinated system behaviour in synthetic microbial consortia, Nature Communications, Vol: 9, Pages: 1-9, ISSN: 2041-1723
Advancing synthetic biology to the multicellular level requires the development of multiple cell-to-cell communication channels that propagate information with minimal signal interference. The development of quorum-sensing devices, the cornerstone technology for building microbial communities with coordinated system behaviour, has largely focused on cognate acyl-homoserine lactone (AHL)/transcription factor pairs, while the use of non-cognate pairs as a design feature has received limited attention. Here, we demonstrate a large library of AHL-receiver devices, with all cognate and non-cognate chemical signal interactions quantified, and we develop a software tool that automatically selects orthogonal communication channels. We use this approach to identify up to four orthogonal channels in silico, and experimentally demonstrate the simultaneous use of three channels in co-culture. The development of multiple non-interfering cell-to-cell communication channels is an enabling step that facilitates the design of synthetic consortia for applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation and spatial organisation.
Freemont PS, Moore S, MacDonald J, et al., 2018, Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from non-model bacteria, Proceedings of the National Academy of Sciences, Vol: 115, Pages: E4340-E4349, ISSN: 0027-8424
Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications.
Tomazou M, Barahona M, Polizzi K, et al., 2018, Computational re-design of synthetic genetic oscillators for independent amplitude and frequency modulation, Cell Systems, Vol: 6, Pages: 508-520.e5, ISSN: 2405-4712
To perform well in biotechnology applications, synthetic genetic oscillators must be engineered to allow independent modulation of amplitude and period. This need is currently unmet. Here, we demonstrate computationally how two classic genetic oscillators, the dual-feedback oscillator and the repressilator, can be re-designed to provide independent control of amplitude and period and improve tunability—that is, a broad dynamic range of periods and amplitudes accessible through the input “dials.” Our approach decouples frequency and amplitude modulation by incorporating an orthogonal “sink module” where the key molecular species are channeled for enzymatic degradation. This sink module maintains fast oscillation cycles while alleviating the translational coupling between the oscillator's transcription factors and output. We characterize the behavior of our re-designed oscillators over a broad range of physiologically reasonable parameters, explain why this facilitates broader function and control, and provide general design principles for building synthetic genetic oscillators that are more precisely controllable.
Elani Y, Trantidou T, Wylie D, et al., 2018, Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules, Scientific Reports, Vol: 8, Pages: 1-8, ISSN: 2045-2322
There is increasing interest in constructing artificial cells by functionalising lipid vesicles with biological and synthetic machinery. Due to their reduced complexity and lack of evolved biochemical pathways, the capabilities of artificial cells are limited in comparison to their biological counterparts. We show that encapsulating living cells in vesicles provides a means for artificial cells to leverage cellular biochemistry, with the encapsulated cells serving organelle-like functions as living modules inside a larger synthetic cell assembly. Using microfluidic technologies to construct such hybrid cellular bionic systems, we demonstrate that the vesicle host and the encapsulated cell operate in concert. The external architecture of the vesicle shields the cell from toxic surroundings, while the cell acts as a bioreactor module that processes encapsulated feedstock which is further processed by a synthetic enzymatic metabolism co-encapsulated in the vesicle.
Jonas FRH, Royle KE, Aw R, et al., 2018, Investigating the consequences of asymmetric endoplasmic reticulum inheritance in Saccharomyces cerevisiae under stress using a combination of single cell measurements and mathematical modelling, Synthetic and Systems Biotechnology, Vol: 3, Pages: 64-75, ISSN: 2405-805X
Adaptation allows organisms to maintain a constant internal environment, which is optimised for growth. The unfolded protein response (UPR) is an example of a feedback loop that maintains endoplasmic reticulum (ER) homeostasis, and is characteristic of how adaptation is often mediated by transcriptional networks. The more recent discovery of asymmetric division in maintaining ER homeostasis, however, is an example of how alternative non-transcriptional pathways can exist, but are overlooked by gold standard transcriptomic or proteomic population-based assays. In this study, we have used a combination of fluorescent reporters, flow cytometry and mathematical modelling to explore the relative roles of asymmetric cell division and the UPR in maintaining ER homeostasis. Under low ER stress, asymmetric division leaves daughter cells with an ER deficiency, necessitating activation of the UPR and prolonged cell cycle during which they can recover ER functionality before growth. Mathematical analysis of and simulation results from our mathematical model reinforce the experimental observations that low ER stress primarily impacts the growth rate of the daughter cells. These results demonstrate the interplay between homeostatic pathways and the importance of exploring sub-population dynamics to understand population adaptation to quantitatively different stresses.
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