Publications
118 results found
Lai H-E, Moore S, Polizzi K, et al., 2018, EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology., Pages: 429-444
Development of advanced synthetic biology tools is always in demand since they act as a platform technology to enable rapid prototyping of biological constructs in a high-throughput manner. EcoFlex is a modular cloning (MoClo) kit for Escherichia coli and is based on the Golden Gate principles, whereby Type IIS restriction enzymes (BsaI, BsmBI, BpiI) are used to construct modular genetic elements (biological parts) in a bottom-up approach. Here, we describe a collection of plasmids that stores various biological parts including promoters, RBSs, terminators, ORFs, and destination vectors, each encoding compatible overhangs allowing hierarchical assembly into single transcription units or a full-length polycistronic operon or biosynthetic pathway. A secondary module cloning site is also available for pathway optimization, in order to limit library size if necessary. Here, we show the utility of EcoFlex using the violacein biosynthesis pathway as an example.
Kylilis N, Stan G-B, Polizzi K, 2017, Tools for engineering coordinated system behaviour in synthetic microbial consortia, Publisher: bioRxiv
Advancing synthetic biology to the multicellular level requires the development of multiple orthogonal cell-to-cell communication channels to 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 reducing signal leakage between systems of cognate AHL/transcription factor pairs. However, the use of non-cognate signals as a design feature has received limited attention so far. Here, we demonstrate the largest library of AHL-receiver devices constructed to date with all cognate and non-cognate chemical signal interactions quantified and we develop a software tool that allows automated selection of orthogonal chemical 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 will facilitate the design of synthetic microbial consortia for novel applications including distributed bio-computation, increased bioprocess efficiency, cell specialisation, and spatial organisation.
Royle KE, Polizzi KM, 2017, A streamlined cloning workflow minimising the time-to-strain pipeline for Pichia pastoris, Scientific Reports, Vol: 7, ISSN: 2045-2322
Although recent advances in E. coli self-assembly have greatly simplified cloning, these have not yet been harnessed for the high-throughput generation of expression strains in the early research and discovery phases of biopharmaceutical production. Here, we have refined the technique and incorporated it into a streamlined workflow for the generation of Pichia pastoris expression strains, reducing the timeline by a third and removing the reliance on DNA editing enzymes, which often require troubleshooting and increase costs. We have validated the workflow by cloning 24 human proteins of biopharmaceutical value, either as direct therapeutics or as research targets, which span a continuous range in size and GC content. This includes demonstrating the applicability of the workflow to three-part assemblies for a monoclonal antibody and its single-chain antibody fragments derivatives. This workflow should enable future research into recombinant protein production by P. pastoris and a synthetic biology approach to this industrial host.
Ogonah OW, Polizzi KM, Bracewell DG, 2017, Cell free protein synthesis: a viable option for stratified medicines manufacturing?, CURRENT OPINION IN CHEMICAL ENGINEERING, Vol: 18, Pages: 77-83, ISSN: 2211-3398
Anastasiadi M, Polizzi K, Lambert RJW, 2017, An improved model for the analysis of combined antimicrobials: a replacement for the Chou-Talalay combination index method, Journal of Applied Microbiology, Vol: 124, Pages: 97-107, ISSN: 1364-5072
AIMS: To rationalise confusion in the literature concerning the analysis of combined antimicrobials, specifically to see if the combination index (CI) method of analysis was as rigorous as claimed. METHODS & RESULTS: data from previous studies of the inhibition of Staphylococcus aureus by mixed antimicrobials were re-analysed using the CI method and a model which takes account of differences in the concentration exponents of individual antimicrobials. CONCLUSIONS: The Chou-Talalay combination index method for the analysis of combined antimicrobials was found to be valid only in the specific cases where concentration exponents were equal. In these cases the CI method was found to be a function of the residuals of fitting the additive model to the observed data. Where concentration exponents were not equal the CI method was invalid, whereas the additive model took these differences into account. SIGNIFICANCE AND IMPACT OF STUDY: The CI method can be replaced wholly by the additive model described. The model allows simple regression to be used to analyse whole data sets and provides simple graphical output. This article is protected by copyright. All rights reserved.
Sou SN, Ken L, Nayyar K, et al., 2017, Exploring cellular behaviour under transient geneexpression and its impact on mAb productivity and Fc glycosylation, Biotechnology and Bioengineering, Vol: 115, Pages: 512-518, ISSN: 1097-0290
Transient gene expression (TGE) is a methodology employed in bioprocessing for the fast provision of recombinant protein material. Mild hypothermia is often introduced to overcome the low yield typically achieved with TGE and improve specific protein productivity. It is therefore of interest to examine the impact of mild hypothermic temperatures on both the yield and quality of transiently-expressed proteins and the relationship to changes in cellular processes and metabolism. In this study, we focus on the ability of a Chinese hamster ovary cell line to galactosylate a recombinant monoclonal antibody (mAb) product. Through experimentation and flux balance analysis, our results show that TGE in mild hypothermic conditions led to a 76% increase in qP compared to TGE at 36.5°C in our system. This increase is accompanied by increased consumption of nutrients and amino acids, together with increased production of intracellular nucleotide sugar species and higher rates of mAb galactosylation, despite a reduced rate of cell growth. The reduction in biomass accumulation allowed cells to redistribute their energy and resources towards mAb synthesis and Fc-glycosylation. Interestingly, the higher capacity of cells to galactosylate the recombinant product in TGE at 32°C appears not to have been assisted by the upregulation of galactosyltransferases (GalTs), but by the increased expression of N-acetylglucosaminyltransferase II (GnTII) in this cell line, which facilitated the production of bi-antennary glycan structures for further processing.
Dekker L, Polizzi KM, 2017, Sense and sensitivity in bioprocessing-detecting cellular metabolites with biosensors., Current Opinion in Chemical Biology, Vol: 40, Pages: 31-36, ISSN: 1367-5931
Biosensors use biological elements to detect or quantify an analyte of interest. In bioprocessing, biosensors are employed to monitor key metabolites. There are two main types: fully biological systems or biological recognition coupled with physical/chemical detection. New developments in chemical biosensors include multiplexed detection using microfluidics. Synthetic biology can be used to engineer new biological biosensors with improved characteristics. Although there have been few biosensors developed for bioprocessing thus far, emerging trends can be applied in the future. A range of new platform technologies will enable rapid engineering of new biosensors based on transcriptional activation, riboswitches, and Förster Resonance Energy Transfer. However, translation to industry remains a challenge and more research into the robustness biosensors at scale is needed.
Goers L, Ainsworth C, Goey CH, et al., 2017, Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production, Biotechnology and Bioengineering, Vol: 114, Pages: 1290-1300, ISSN: 1097-0290
Many high-value added recombinant proteins, such as therapeutic glycoproteins, are produced using mammalian cell cultures. In order to optimise the productivity of these cultures it is important to monitor cellular metabolism, for example the utilisation of nutrients and the accumulation of metabolic waste products. One metabolic waste product of interest is lactic acid (lactate), overaccumulation of which can decrease cellular growth and protein production. Current methods for the detection of lactate are limited in terms of cost, sensitivity, and robustness. Therefore, we developed a whole-cell Escherichia coli lactate biosensor based on the lldPRD operon and successfully used it to monitor lactate concentration in mammalian cell cultures. Using real samples and analytical validation we demonstrate that our biosensor can be used for absolute quantification of metabolites in complex samples with high accuracy, sensitivity and robustness. Importantly, our whole-cell biosensor was able to detect lactate at concentrations more than two orders of magnitude lower than the industry standard method, making it useful for monitoring lactate concentrations in early phase culture. Given the importance of lactate in a variety of both industrial and clinical contexts we anticipate that our whole-cell biosensor can be used to address a range of interesting biological questions. It also serves as a blueprint for how to capitalise on the wealth of genetic operons for metabolite sensing available in Nature for the development of other whole-cell biosensors.
Goers L, Ainsworth C, Goey CH, et al., 2017, Cover Image, Volume 114, Number 6, June 2017.
Cover Legend The cover image, by Lisa Goers et al., is based on the Article Whole-cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production, DOI: 10.1002/bit.26254.
Walker BJ, stan GB, Polizzi KM, 2017, Intracellular delivery of biologic therapeutics by bacterial secretion systems, Expert Reviews in Molecular Medicine, Vol: 19, ISSN: 1462-3994
Biologics are a promising new class of drugs based on complex macromolecules such as proteins and nucleic acids. However, delivery of these macromolecules into the cytoplasm of target cells remains a significant challenge. Here we present one potential solution: bacterial nanomachines that have evolved over millions of years to efficiently deliver proteins and nucleic acids across cell membranes and between cells. In this review, we provide a brief overview of the different bacterial systems capable of direct delivery into the eukaryotic cytoplasm and the medical applications for which they are being investigated, along with a perspective on the future directions of this exciting field.
Aw, McKay, Shattock, et al., 2017, Expressing anti-HIV VRC01 antibody using the murine IgG1 secretion signal in Pichia pastoris, AMB Express, Vol: 7, ISSN: 2191-0855
The use of the recombinant expression platform Pichia pastoris to produce pharmaceutically important proteins has been investigated over the past 30 years. Compared to mammalian cultures, expression in P. pastoris is cheaper and faster, potentially leading to decreased costs and process development times. Product yields depend on a number of factors including the secretion signal chosen for expression, which can influence the host cell response to recombinant protein production. VRC01, a broadly neutralising anti-HIV antibody, was expressed in P. pastoris, using the methanol inducible AOX1 promoter for both the heavy and light chains. Titre reached up to 3.05 μg mL-1 in small scale expression. VRC01 was expressed using both the α-mating factor signal peptide from Saccharomyces cerevisiae and the murine IgG1 signal peptide. Surprisingly using the murine IgG1 signal peptide resulted in higher yield of antibody capable of binding gp140 antigen. Furthermore, we evaluated levels of secretory stress compared to the untransformed wild-type strain and show a reduced level of secretory stress in the murine IgG1 signal peptide strains versus those containing the α-MF signal peptide. As bottlenecks in the secretory pathway are often the limiting factor in protein secretion, reduced levels of secretory stress and the higher yield of functional antibody suggest the murine IgG1 signal peptide may lead to better protein folding and secretion. This work indicates the possibilities for utilising the murine IgG1 signal peptide for a range of antibodies, resulting in high yields and reduced cellular stress.
Sou SN, Jedrzejewski PM, Lee K, et al., 2017, Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia, Biotechnology and Bioengineering, Vol: 114, Pages: 1570-1582, ISSN: 1097-0290
Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (qmAb) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc-glycosylation and the mechanism behind changes in the glycan composition is not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimisation. To address this issue, a mathematical model that quantitatively describes CHO cell behaviour and metabolism, mAb synthesis and its N-linked glycosylation profiles before and after the induction of mild hypothermia is constructed using two sets of parameters. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N-linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggest the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc-glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimisation, e.g., by tailoring feeding stradegies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering.
Moore SJ, Lai HE, Needham H, et al., 2017, Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool, Biotechnology Journal, Vol: 12, ISSN: 1860-7314
Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
Moore SJ, lai H-E, Kelwick R, et al., 2016, EcoFlex - a multifunctional MoClo kit for E. coli synthetic biology, ACS Synthetic Biology, Vol: 5, Pages: 1059-1069, ISSN: 2161-5063
Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimisation, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli, has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterise in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimise pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimisation. In summary, EcoFlex provides a standardised and multifunctional kit for a variety of applications in E. coli synthetic biology.
Klymenko O, Royle K, Polizzi KM, et al., 2016, Designing an Artificial Golgi Reactor to achieve targeted glycosylation of monoclonal antibodies, AICHE Journal, Vol: 62, Pages: 2959-2973, ISSN: 0001-1541
The therapeutic efficacy of monoclonal antibodies (mAbs) is dependent upon their glycosylationpatterns. As the largest group of currently approved biopharmaceuticals, the microheterogeneity inmAb oligosaccharide profiles deriving from mammalian cell production is a challenge to thebiopharmaceutical industry. Disengaging the glycosylation process from the cell may offer significantenhancement of product quality and allow better control and reproducibility in line with the Quality byDesign paradigm. Three potential designs of an Artificial Golgi reactor implementing targeted sequentialglycosylation of mAbs are proposed including a (i) microcapillary film reactor, (ii) packed bed reactorwith non-porous pellets, and (iii) packed bed reactor with porous pellets. Detailed mathematical modelsare developed to predict their performance for a range of design and operational parameters. While allthree reactor designs can achieve desired conversion levels, the choice of a particular one depends onthe required throughput and the associated cost of enzymes and co-substrates.
Jimenez del Val I, Polizzi K, Kontoravdi C, 2016, A theoretical estimate for nucleotide sugar demand towards Chinese Hamster Ovary cellular glycosylation, Scientific Reports, Vol: 6, ISSN: 2045-2322
Glycosylation greatly influences the safety and efficacy of many of the highest-selling recombinant therapeutic proteins (rTPs). In order to define optimal cell culture feeding strategies that control rTP glycosylation, it is necessary to know how nucleotide sugars (NSs) are consumed towards host cell and rTP glycosylation. Here, we present a theoretical framework that integrates the reported glycoproteome of CHO cells, the number of N-linked and O-GalNAc glycosylation sites on individual host cell proteins (HCPs), and the carbohydrate content of CHO glycosphingolipids to estimate the demand of NSs towards CHO cell glycosylation. We have identified the most abundant N-linked and O-GalNAc CHO glycoproteins, obtained the weighted frequency of N-linked and O-GalNAc glycosites across the CHO cell proteome, and have derived stoichiometric coefficients for NS consumption towards CHO cell glycosylation. By combining the obtained stoichiometric coefficients with previously reported data for specific growth and productivity of CHO cells, we observe that the demand of NSs towards glycosylation is significant and, thus, is required to better understand the burden of glycosylation on cellular metabolism. The estimated demand of NSs towards CHO cell glycosylation can be used to rationally design feeding strategies that ensure optimal and consistent rTP glycosylation.
Aw R, Polizzi KM, 2016, Liquid PTVA: A faster and cheaper alternative for generating multi-copy clones in Pichia pastoris, Microbial Cell Factories, Vol: 15, ISSN: 1475-2859
BACKGROUND:Multiple cognate gene copy clones have often been used in order to increase the yield of recombinant protein expression in the yeast Pichia pastoris. The method of posttransformational vector amplification (PTVA) has allowed for the efficient generation of multi-copy clones in P. pastoris. However, despite its relative ease and success, this process can be expensive and time consuming.RESULTS:We have developed a modified version of PTVA, called Liquid PTVA, which allows for faster and cheaper selection of multi-copy clones. Cultures are grown in liquid medium with only a final selection carried out on agar plates, reducing overall antibiotic usage and increasing the speed of clone amplification. In addition, it was established that starting PTVA with a single copy clone resulted in higher copy number strains for both traditional plate PTVA and liquid PTVA. Furthermore, using the Zeocin selection marker in liquid PTVA results in strains with higher growth rates, which could be beneficial for recombinant protein production processes.CONCLUSIONS:We present a methodology for creating multi-copy clones that can be achieved over 12 days instead of the traditional 45 and at approximately half the cost.
Polizzi KM, Freemont PS, 2016, Synthetic biology biosensors for healthcare and industrial biotechnology applications
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Sou SN, Sellick C, Lee K, et al., 2015, How does mild hypothermia affect monoclonal antibody glycosylation?, Biotechnology and Bioengineering, Vol: 112, Pages: 1165-1176, ISSN: 1097-0290
Baldwin G, Bayer T, Dickinson R, et al., 2015, Synthetic biology - a primer, ISBN: 9781783268801
Synthetic Biology - A Primer (Revised Edition) presents an updated overview of the field of synthetic biology and the foundational concepts on which it is built. This revised edition includes new literature references, working and updated URL links, plus some new figures and text where progress in the field has been made. The book introduces readers to fundamental concepts in molecular biology and engineering and then explores the two major themes for synthetic biology, namely ‘bottom-up’ and ‘top-down’ engineering approaches. ‘Top-down’ engineering uses a conceptual framework of systematic design and engineering principles focused around the Design-Build-Test cycle and mathematical modelling. The ‘bottom-up’ approach involves the design and building of synthetic protocells using basic chemical and biochemical building blocks from scratch exploring the fundamental basis of living systems. Examples of cutting-edge applications designed using synthetic biology principles are presented, including: the production of novel, microbial synthesis of pharmaceuticals and fine chemicals the design and implementation of biosensors to detect infections and environmental waste. The book also describes the Internationally Genetically Engineered Machine (iGEM) competition, which brings together students and young researchers from around the world to carry out summer projects in synthetic biology. Finally, the primer includes a chapter on the ethical, legal and societal issues surrounding synthetic biology, illustrating the integration of social sciences into synthetic biology research. Final year undergraduates, postgraduates and established researchers interested in learning about the interdisciplinary field of synthetic biology will benefit from this up-to-date primer on synthetic biology.
Polizzi KM, Kontoravdi C, 2014, Genetically-encoded biosensors for monitoring cellular stress in bioprocessing, Current Opinion in Biotechnology, Vol: 31, Pages: 50-56, ISSN: 1879-0429
Goers L, Freemont P, Polizzi KM, 2014, Co-culture systems and technologies: taking synthetic biology to the next level, JOURNAL OF THE ROYAL SOCIETY INTERFACE, Vol: 11, ISSN: 1742-5689
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- Citations: 345
Jedrzejewski PM, Jimenez del Val I, Constantinou A, et al., 2014, Towards controlling the glycoform: a model framework linking extracellular metabolites to antibody glycosylation, International Journal of Molecular Sciences, Vol: 15, Pages: 4492-4522, ISSN: 1422-0067
Glycoproteins represent the largest group of the growing number of biologically-derived medicines. The associated glycan structures and their distribution are known to have a large impact on pharmacokinetics. A modelling framework was developed to provide a link from the extracellular environment and its effect on intracellular metabolites to the distribution of glycans on the constant region of an antibody product. The main focus of this work is the mechanistic in silico reconstruction of the nucleotide sugar donor (NSD) metabolic network by means of 34 species mass balances and the saturation kinetics rates of the 60 metabolic reactions involved. NSDs are the co-substrates of the glycosylation process in the Golgi apparatus and their simulated dynamic intracellular concentration profiles were linked to an existing model describing the distribution of N-linked glycan structures of the antibody constant region. The modelling framework also describes the growth dynamics of the cell population by means of modified Monod kinetics. Simulation results match well to experimental data from a murine hybridoma cell line. The result is a modelling platform which is able to describe the product glycoform based on extracellular conditions. It represents a first step towards the in silico prediction of the glycoform of a biotherapeutic and provides a platform for the optimisation of bioprocess conditions with respect to product quality.
Aw R, Polizzi KM, 2013, Can too many copies spoil the broth?, Microbial Cell Factories, Vol: 12, ISSN: 1475-2859
The success of Pichia pastoris as a heterologous expression system lies predominantly in the impressive yields thatcan be achieved due to high volumetric productivity. However, low specific productivity still inhibits the potentialsuccess of this platform. Multi-(gene) copy clones are potentially a quick and convenient method to increaserecombinant protein titer, yet they are not without their pitfalls. It has been more than twenty years since the firstreported use of multi-copy clones and it is still an active area of research to find the fastest and most efficientmethod for generating these strains. It has also become apparent that there is not always a linear correlationbetween copy number and protein titer, leading to in-depth investigations into how to minimize the negativeimpact of secretory stress and achieve clonal stability.
Behjousiar A, Constantinou A, Polizzi KM, et al., 2013, FIBS-enabled non-invasive metabolic profiling, Journal of Visualised Experiments (accepted)
A description of how to calibrate Förster Resonance Energy Transfer integrated biological sensors (FIBS) for in situ metabolic profiling is presented. The FIBS can be used to estimate intracellular metabolite concentrations non-invasively aiding in the development of metabolic models and high throughput screening of bioprocess conditions.
Constantinou A, Polizzi KM, 2013, Opportunities for bioprocess monitoring using FRET biosensors, BIOCHEMICAL SOCIETY TRANSACTIONS, Vol: 41, Pages: 1146-1151, ISSN: 0300-5127
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- Citations: 15
Kyriakopoulos S, Polizzi KM, Kontoravdi C, 2013, Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity, Journal of Biotechnology, Vol: 168, Pages: 543-551
Jimenez del Val I, Kyriakopoulos S, Polizzi KM, et al., 2013, An optimised method for extraction and quantification of nucleotides and nucleotide sugars from mammalian cells, Analytical Biochemistry, Vol: 443, Pages: 172-180
Arpino JAJ, Hancock EJ, Anderson J, et al., 2013, Tuning the dials of Synthetic Biology, Microbiology-Sgm, Vol: 159, Pages: 1236-1253, ISSN: 1465-2080
Polizzi KM, Constantinou A, Goers L, 2013, Designing metabolite biosensors for bioprocess monitoring using synthetic biology
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