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• Journal article
  Elani Y, Law R, Ces O,

  Vesicle-based artificial cells: recent developments and prospects for drug delivery

  , Therapeutic delivery, ISSN: 2041-6008
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• Journal article
  Campeotto I, Zhang Y, Mladenov MG, Freemont PS, Grundling Aet al., 2015,

  Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family

  , Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351X

  Signaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.

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• Conference paper
  Bricio C, Liu X, Li X, Rehak M, Ellis T, Smith Cet al., 2015,

  High-throughput picodroplet-based analysis of biosynthetic libraries

  , Pages: 709-710
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• Journal article
  Lu M, Williamson N, Boschetti C, Ellis T, Yoshimi T, Tunnacliffe Aet al., 2015,

  Expression-level dependent perturbation of cell proteostasis and nuclear morphology by aggregation-prone polyglutamine proteins

  , Biotechnol. Bioeng., Pages: n/a-n/a, ISSN: 1097-0290

  We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to ∼80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs. This article is protected by copyright. All rights reserved

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• Journal article
  Pothoulakis G, Ellis T, 2015,

  Using Spinach Aptamer to Correlate mRNA and Protein Levels in <i>Escherichia coli</i>

  , RIBOSWITCHES AS TARGETS AND TOOLS, Vol: 550, Pages: 173-185, ISSN: 0076-6879
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  • Citations: 3
• Journal article
  Pan W, Yuan Y, Ljung L, Goncalves J, Stan G-Bet al., 2015,

  Identifying Biochemical Reaction Networks From Heterogeneous Datasets

  , 2015 54TH IEEE CONFERENCE ON DECISION AND CONTROL (CDC), Pages: 2525-2530, ISSN: 0743-1546
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  • Citations: 7
• Journal article
  Kelwick R, MacDonald JT, Webb AJ, Freemont Pet al., 2014,

  Developments in the Tools and Methodologies of Synthetic Biology

  , Frontiers in Bioengineering and Biotechnology, Vol: 2
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• Journal article
  Foerster A, Planamente S, Manoli E, Lossi NS, Freemont PS, Filloux Aet al., 2014,

  Coevolution of the ATPase ClpV, the Sheath Proteins TssB and TssC, and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes

  , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289
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  • Citations: 43
• Journal article
  Robinson T, Valluri P, Kennedy G, Sardini A, Dunsby C, Neil MAA, Baldwin GS, French PMW, de Mello AJet al., 2014,

  Analysis of DNA Binding and Nucleotide Flipping Kinetics Using Two-Color Two-Photon Fluorescence Lifetime Imaging Microscopy

  , Analytical Chemistry, Vol: 86, Pages: 10732-10740, ISSN: 0003-2700

  Uracil DNA glycosylase plays a key role in DNA maintenance via base excision repair. Its role is to bind to DNA, locate unwanted uracil, and remove it using a base flipping mechanism. To date, kinetic analysis of this complex process has been achieved using stopped-flow analysis but, due to limitations in instrumental dead-times, discrimination of the “binding” and “base flipping” steps is compromised. Herein we present a novel approach for analyzing base flipping using a microfluidic mixer and two-color two-photon (2c2p) fluorescence lifetime imaging microscopy (FLIM). We demonstrate that 2c2p FLIM can simultaneously monitor binding and base flipping kinetics within the continuous flow microfluidic mixer, with results showing good agreement with computational fluid dynamics simulations.

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• Journal article
  Campeotto I, Percy MG, MacDonald JT, Foerster A, Freemont PS, Gruendling Aet al., 2014,

  Structural and Mechanistic Insight into the Listeria monocytogenes Two-enzyme Lipoteichoic Acid Synthesis System

  , Journal of Biological Chemistry, Vol: 289, Pages: 28054-28069, ISSN: 0021-9258

  Lipoteichoic acid (LTA) is an important cell wall componentrequired for proper cell growth in many Gram-positive bacteria.In Listeria monocytogenes, two enzymes are required for the synthesisof this polyglycerolphosphate polymer. The LTA primaseLtaPLm initiates LTA synthesis by transferring the first glycerolphosphate(GroP) subunit onto the glycolipid anchor and theLTA synthase LtaSLm extends the polymer by the repeated additionof GroP subunits to the tip of the growing chain. Here, wepresent the crystal structures of the enzymatic domains ofLtaPLm and LtaSLm. Although the enzymes share the same fold,substantial differences in the cavity of the catalytic site andsurface charge distribution contribute to enzyme specialization.The eLtaSLm structure was also determined in complexwith GroP revealing a second GroP binding site. Mutationalanalysis confirmed an essential function for this binding siteand allowed us to propose a model for the binding of thegrowing chain.

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• Journal article
  Rivadeneira PS, Moog CH, Stan G-B, Brunet C, Raffi F, Ferré V, Costanza V, Mhawej MJ, Biafore F, Ouattara DA, Ernst D, Fonteneau R, Xia Xet al., 2014,

  Mathematical Modeling of HIV Dynamics After Antiretroviral Therapy Initiation: A Review.

  , Biores Open Access, Vol: 3, Pages: 233-241, ISSN: 2164-7844

  This review shows the potential ground-breaking impact that mathematical tools may have in the analysis and the understanding of the HIV dynamics. In the first part, early diagnosis of immunological failure is inferred from the estimation of certain parameters of a mathematical model of the HIV infection dynamics. This method is supported by clinical research results from an original clinical trial: data just after 1 month following therapy initiation are used to carry out the model identification. The diagnosis is shown to be consistent with results from monitoring of the patients after 6 months. In the second part of this review, prospective research results are given for the design of individual anti-HIV treatments optimizing the recovery of the immune system and minimizing side effects. In this respect, two methods are discussed. The first one combines HIV population dynamics with pharmacokinetics and pharmacodynamics models to generate drug treatments using impulsive control systems. The second one is based on optimal control theory and uses a recently published differential equation to model the side effects produced by highly active antiretroviral therapy therapies. The main advantage of these revisited methods is that the drug treatment is computed directly in amounts of drugs, which is easier to interpret by physicians and patients.

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• Journal article
  Jovicevic D, Blount BA, Ellis T, 2014,

  Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast

  , BIOESSAYS, Vol: 36, Pages: 855-860, ISSN: 0265-9247
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  • Citations: 23
• Journal article
  Rivadeneira PS, Moog CH, Stan G-B, Costanza V, Brunet C, Raffi F, Ferre V, Mhawej M-J, Biafore F, Ouattara DA, Ernst D, Fonteneau R, Xia Xet al., 2014,

  Mathematical Modeling of HIV Dynamics After Antiretroviral Therapy Initiation: A Clinical Research Study

  , AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol: 30, Pages: 831-834, ISSN: 0889-2229
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  • Citations: 8
• Conference paper
  Polizzi KM, Kylilis N, Lai HE, Freemont PSet al., 2014,

  Detecting protein biomarkers using engineered biosensors based on synthetic biology principles

  , 248th National Meeting of the American-Chemical-Society (ACS), Publisher: AMER CHEMICAL SOC, ISSN: 0065-7727
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• Journal article
  Casini A, Christodoulou G, Freemont PS, Baldwin GS, Ellis T, MacDonald JTet al., 2014,

  R2oDNA Designer: Computational Design of Biologically Neutral Synthetic DNA Sequences

  , ACS SYNTHETIC BIOLOGY, Vol: 3, Pages: 525-528, ISSN: 2161-5063
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  • Citations: 47
• Journal article
  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
• Journal article
  Galdzicki M, Clancy KP, Oberortner E, Pocock M, Quinn JY, Rodriguez CA, Roehner N, Wilson ML, Adam L, Anderson JC, Bartley BA, Beal J, Chandran D, Chen J, Densmore D, Endy D, Gruenberg R, Hallinan J, Hillson NJ, Johnson JD, Kuchinsky A, Lux M, Misirli G, Peccoud J, Plahar HA, Sirin E, Stan G-B, Villalobos A, Wipat A, Gennari JH, Myers CJ, Sauro HMet al., 2014,

  The Synthetic Biology Open Language (SBOL) provides a community standard for communicating designs in synthetic biology

  , NATURE BIOTECHNOLOGY, Vol: 32, Pages: 545-550, ISSN: 1087-0156
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  • Citations: 182
• Journal article
  Oyarzun DA, Lugagne J-B, Stan G-B, 2014,

  Noise propagation in synthetic gene circuits for metabolic control

  , ACS Synthetic Biology, Vol: 4, Pages: 116-125, ISSN: 2161-5063

  Dynamic control of enzyme expression can be an effective strategy to engineer robust metabolic pathways. It allows a synthetic pathway to self-regulate in response to changes in bioreactor conditions or the metabolic state of the host. The implementation of this regulatory strategy requires gene circuits that couple metabolic signals with the genetic machinery, which is known to be noisy and one of the main sources of cell-to-cell variability. One of the unexplored design aspects of these circuits is the propagation of biochemical noise between enzyme expression and pathway activity. In this article, we quantify the impact of a synthetic feedback circuit on the noise in a metabolic product in order to propose design criteria to reduce cell-to-cell variability. We consider a stochastic model of a catalytic reaction under negative feedback from the product to enzyme expression. On the basis of stochastic simulations and analysis, we show that, depending on the repression strength and promoter strength, transcriptional repression of enzyme expression can amplify or attenuate the noise in the number of product molecules. We obtain analytic estimates for the metabolic noise as a function of the model parameters and show that noise amplification/attenuation is a structural property of the model. We derive an analytic condition on the parameters that lead to attenuation of metabolic noise, suggesting that a higher promoter sensitivity enlarges the parameter design space. In the theoretical case of a switch-like promoter, our analysis reveals that the ability of the circuit to attenuate noise is subject to a trade-off between the repression strength and promoter strength.

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• Journal article
  Ewens CA, Panico S, Kloppsteck P, McKeown C, Ebong I-O, Robinson C, Zhang X, Freemont PSet al., 2014,

  The p97-FAF1 Protein Complex Reveals a Common Mode of p97 Adaptor Binding

  , JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 289, Pages: 12077-12084
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  • Citations: 20
• Journal article
  Pothoulakis G, Ceroni F, Reeve B, Ellis Tet 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.

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