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Bioenergy and Industrial Biotechnology (IBBE) Rotation Projects 2014-2015

Computer-aided design of efficient bacterial promoters

Supervisor: Dr. Boris Adryan, Genetics

Project abstract: On top of our project advertised as part of the "World Class Underpinning Bioscience/New Ways of Working" programme, some of our work on gene regulation control may have implications for Industrial Biotechnology. Students interested in these topics should have a look at our website, particularly item 4 on http://logic.sysbiol.cam.ac.uk/?page_id=27.    Our work on physical constraints of bacterial promoter architecture suggests that the response (e.g., dynamic response vs stable expression) can be influenced by transcription factor specificity, transcription factor concentration, and spacing between the binding sites. Previous work in our lab concentrated on the description of the promoter repertoire in E. coli. However, a student with affinity to computational methods could explore how bacterial promoters can be "optimised" to obtain particular features.

Learning outcomes and skills acquired: Learning outcome: Deep understanding of the physical  constraints that influence transcription factor target finding and gene regulation.  Skills: How to initialise and run stochastic simulations of transcription factor target finding, and how to analyse the numerical output of these programs to infer biological principles.

Project availability: Michaelmas and Lent Term

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Use of microdroplets for selection in algal synthetic biology

Supervisor: Professor Chris Abell, Chemistry

Second supervisor: Professor Alison Smith, Plant Sciences

Project abstract: Microdroplets are small (nano to picoliter) water droplets constrained within a microfluidic device. Each one is a potential reaction vessel in which individual cells can be isolated and monitored.  Droplets can be generated and monitored a rates above 10,000 per second, allowing very high throughput analysis, whilst using very little material. We use these droplets for studying processes at the single cell level. A particular focus is in the area of algal synthetic biology. We are looking to generate organisms that have enhanced characteristics e.g. amount or quality of lipid produced.  The microdroplets platform is then used to select clones of interest and to study their growth characteristics. We are particularly interested in stochastic variation within a clonal population.  The project draws on the respective expertise in the Abell group (microdroplets) and the Smith group (algal biology).

Learning outcomes and skills acquired: The placement will provide a technical introduction to the use of the microdroplet platform.  This involves learning how to assemble devices, load algal cells into droplets, and study them through an algal growth cycle. Training will be given in the use of lasers, and the collection and analysis of fluorescent signals from droplets.   The advantages and drawbacks of studying processes at the single cell level will be explored.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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In-vitro simulation of major physiological barriers

Supervisor: Professor Nigel Slater, Chemical Engineering and Biotechnology

Project abstract: Drug delivery for certain diseases is hampered by the difficulty of transferring biologically active materials across certain physiological barriers, notably the blood-brain barrier, the air-blood barrier in the lungs and the lining of the intestinal tract.  This short project will involve the co-cultivation of two different cell lines on either side of a synthetic membrane in order to provide an in-vitro simulation system that will be used to study the transport behaviour of novel nanoparticle drug delivery systems.  The student would work alongside other researchers in Cambridge involved in an EU-ITN project on nanoparticle-membrane interactions, who are designing new drug delivery materials, and in consultation with researches at the University Medical Centre in Mainz, who have established such in-vitro co-culture systems.  Training will be provided in cell culture and the imaging of surrogate drug molecular transport into cells and across the in-vitro cell barriers.

Learning outcomes and skills acquired: -    Ability to cultivate mammalian cells and monitor physiological parameters relevant to growth.  -        Ability to image molecular transport into cells and across cell barriers.  -       An understanding of the design and function of nanoparticle drug delivery vehicles.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Enzymatic release of sugars from plant biomass

Supervisor: Professor Paul Dupree, Biochemistry

Project abstract: Despite the fact that the plant cell wall forms one of the largest biomasses on Earth and is an important component of food and energy, many fundamental aspects of its structure and function, and the enzymes responsible for its synthesis are still largely a mystery. The aim of our research is to understand how to improve plants and enzymes for increased sugar release from biomass for food and energy.   Cellulose and xylan cannot be easily degraded because of poorly understood aspects – both of their structure, and of their environment in the cell walls. We have generated a number of mutants in cell wall biosynthesis, which lead to alterations in cell wall composition, such as lignin structure and quantity, or xylan structure and quantity. This project will use these mutants to test the hypothesis that these cell wall components prevent access of cell wall degrading enzymes to their substrates, such as cellulose. 

Bromley et al. (2013) Plant J. 74(3):423–434. 

Quinlan et al. (2011) PNAS 108(37):15079-84.

Learning outcomes and skills acquired: The student will learn plant growth, plant cell wall preparation, hydrolase enzyme digestion of polysaccharides to determine structures, enzyme characaterisation, and PACE- gel electrophoresis of oligosaccharides.

Project availability: Michaelmas and Lent Term

Other relevant themes: Food security

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Genome mining of antibiotic natural product biosynthetic gene clusters

Supervisor: Professor Peter Leadlay FRS, Biochemistry

Project abstract: We have developed rapid methods, using our in-house nextgeneration sequencing facility, for the essentially complete sequencing and assembly of the 8-12 Mbp linear genomes of Streptomyces and allied Gram-positive filamentous bacteria. Such bacteria have produced approximately 70% of all antibiotics identified to date. The sequencing has revealed genomic islands in each genome predicted to contain a total of between 20 and 40 biosynthetic gene clusters. An urgent task now is to link each gene cluster to its chemical product. In this project, one such genome will be explored for its "diversome" by growing the strain under various conditions on solid and liquid media and examining the HPLC-MS profiles. The genome will be selected on the basis that it is known to produce at least one known antibiotic whose biosynthetic pathway is unexplored. Some clusters will be more predictable than others, aided by tools such as Antismash 2.0 (http://www.secondarymetabolites.org/) which provides a first-pass census of clusters in a genome. By definition, though, to detect novel clusters will require close manual scrutiny. One or two clusters will be selected for genetic knockout experiments to confirm the link between gene cluster and chemical product.

Learning outcomes and skills acquired: The techniques to be used include: genome analysis software; mainstream molecular microbiology; recombinant DNA techniques for filamentous streptomycetes; natural product identification and structure determination.

Project availability: Michaelmas and Lent Term

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The structure and function of assembly-line antibiotic synthases: detection of assembly intermediates on multienzyme polyketide synthases using a novel trapping method.

Supervisor: Professor Peter Leadlay FRS, Biochemistry

Project abstract: In conjunction with Dr Manuela Tosin (U of Warwick) we have developed a versatile and convenient way of capturing intermediates in vivo from a biosynthetic assembly-line and identifying them by mass spectrometric analysis. This is revealing remarkable insights into the timing of enzymatic steps, and helping lay the foundation for engineering of these remarkable systems to produce novel versions of potentially valuable pharmaceuticals and agrochemicals. In this project, we will study the intermediates in the assembly of the polyether premonensin, a model system for polyketide synthase mechanism; and of the antifungal azalomycin, which appears to follow a non-canonical mechanism. Such exceptions to the modular rule promise to provide important clues to the natural evolution of modular assembly-line catalysts.     References:  Riva, E. Wilkening, I., Gazzola, S., Li, A., Smith, L., Leadlay, P. F., Tosin, M., (2014) Chemical probes for the functionalization of polyketide intermediates: towards novel 'unnatural' products. Angew. Chem. Int. Ed. Engl. under revision. (to be highlighted by Angewandte Chemie  as a “VIP” paper).  Tosin, M., Smith, L., Leadlay, P. F. (2011) Insights into lasalocid ring formation via chemical chain termination in vivo. Angew. Chem. Int. Ed. Engl. 50, 11930-11933. (highlighted by Angewandte Chemie  as a “VIP” paper).

Learning outcomes and skills acquired: Techniques to be used include: mainstream molecular microbiology; recombinant DNA techniques for filamentous streptomycetes; natural product isolation and structure determination using high resolution mass spectrometers.

Project availability: Michaelmas and Lent Term

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Biophysical characterization of a polyketide synthase module

Supervisor: Dr. Bill Broadhurst, Biochemistry

Project abstract: This project aims to scale up the expression and purification of an intact polyketide synthase module from the multienzyme complex responsible for making the broad-spectrum antibiotic azalomycin. Following this, a range of biophysical methods will be used to characterize the protein in vitro, including: mass spectrometry-based functional assays; size exclusion chromatography, dynamic light scattering and analytical ultracentrifugation to define the oligomeric state; circular dichroism spectroscopy to assess the secondary structure; hydrogen/deuterium exchange experiments to identify buried protein-protein interfaces; and cysteine mutagenesis followed by covalent attachment of dyes to facilitate studies by fluorescence spectroscopy.

Learning outcomes and skills acquired: The student will gain experience in standard molecular biology methods, including: transformation and expression of bacterial proteins in E. coli cells; site directed mutagenesis; strategies for optimising expression of soluble protein; protein purification using affinity, size exclusion and ion exchange chromatography; use of a liquid chromatography-mass spectrometer for enzyme product identification; use of a plate reader for colorimetric and fluorescence based functional assays; and use of dynamic light scattering, circular dichroism and analytical ultracentrifugation equipment.

Project availability: Michaelmas and Lent Term

Other relevant themes: World class underpinning bioscience

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Solution structure of a loading module acyl carrier protein domain

Supervisor: Dr. Bill Broadhurst, Biochemistry

Project abstract: The aim of this project is to determine the solution structure of a small (90 amino acid) protein domain from the loading module of the polyketide synthase responsible for producing the antibiotic erythromycin, which is widely used to combat respiratory tract infections. We have already defined expression and purification protocols for the acyl carrier protein domain (ACP0), and have completed the assignment of resonances from backbone nuclei using nuclear magnetic resonance spectroscopy. The next steps are to prepare a sample that is uniformly labelled with 15N and 13C stable isotopes, collect three dimensional side-chain assignment and NOESY spectra, catalog the distance restraints and then perform structure calculations using the ARIA/CNS package. Structural information on ACP0 will provide a firm foundation for characterizing its mode of interaction with the loading acyltransferase domain and with the ketosynthase domain of the first chain-extension module. Knowledge of this sort is a crucial prerequisite for “combinatorial biosynthesis” strategies to engineer polyketide synthase systems for efficient generation of novel molecular scaffolds that can subsequently be screened for biological activity.

Learning outcomes and skills acquired: The student will gain experience in standard molecular biology methods, including expression of bacterial proteins in minimal media and subsequent purification using affinity and size exclusion chromatography. Next, the student will be partnered with an expert spectroscopist to collect the NMR experiments, and will then use a state-of-the-art software package (CCPNMR Analysis) to process and analyse the data. Structure calculations will be carried out using the ARIA/CNS package, which can interpret “ambiguous” (partially assigned) distance restraints and employs many up-to-date structure refinement protocols. This experience will provide a robust introduction to current structural biology methods.

Project availability: Michaelmas and Lent Term

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Photosynthetic Organisms in Biotechnology and Health

Supervisor: Prof Chris Howe, Biochemistry

Project abstract: Projects are available in a number of aspects of the molecular biology of photosynthetic organisms and their exploitation, including the (no longer photosynthetic) malaria parasite Plasmodium.     Specific areas include:    (i) Direct electricity production from photosynthetic micro-organisms.  Surprisingly, cyanobacterial and eukaryotic algal cells produce a small amount of external current on illumination, which can be harvested by an anode and used to drive an external circuit. We are interested in understanding how to make these ‘biophotovoltaic’ devices more efficient, and to drive additional processes, such as hydrogen production. We are also developing similar devices using land plants.    (ii) Manipulation of photosynthetic microorganisms, including cyanobacteria and purple photosynthetic bacteria, for production of useful products. This includes work on light harvesting, electron transfer (including a novel c-type cytochrome we discovered) and metabolic engineering (including expressing heterologous genes and modifying endogenous ones). We have a wide range of molecular genetic platforms available for this. We are also trying to develop dinoflagellate algae as a biotechnology platform.    (iii) Novel antimalarial targets. Surprisingly, the malaria parasite Plasmodium has a photosynthetic ancestry and a remnant chloroplast, which is essential for the parasite to survive. This offers an attractive target for antimalarials. We are focusing on transcription of the remnant chloroplast genome and post-transcriptional RNA processing. We have identified a number of candidate nuclear genes for chloroplast proteins that are likely to be involved in these processes.    See the Howe group web site (http://www.bioc.cam.ac.uk/howe) for further information.

Learning outcomes and skills acquired: Depending on the particular project, expertise in:    Handling photosynthetic microorganisms (cyanobacteria, algae, dinoflagellates) and Plasmodium.  Routine molecular biology.  Electrochemistry and biophotovoltaics.  Renewable energy generation and algal biotechnology.  Photosynthesis biochemistry and molecular evolution.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Integration of Photosynthetic Proteins in Nanostructured Devices for the Generation of Solar Fuels

Supervisor: Dr. Erwin Reisner, Chemistry

Second supervisor:  

Project abstract: We address the technological and scientific challenge of the generation of renewable fuels such as hydrogen and carbon-feedstocks by using sunlight as energy input and isolated photosynthetic enzymes offered from biology as catalysts. We will adopt the principles of natural photosynthesis and components that are part of this fundamental biological process to use solar energy for the production of renewable fuels. Direct conversion of solar energy to a fuel is important, because the majority of the worlds energy is used in the form of a fuel, and not electricity.   The project will be in line with previous achievements of the Reisner group on solar fuel generation with enzyme-hybrid systems. Visible light driven reduction of aqueous protons to H2,[1] and conversion of CO2 to CO[2] was achieved with enzyme-modified dye-sensitised TiO2 nanoparticles. In this system, direct solar fuel generation was achieved by making use of dye-TiO2, which is the best part of dye-sensitised photovoltaic cells for visible light harvesting and efficient charge separation. As enzymes we employed [NiFe]-hydrogenase (to reduce protons) and NiFe-containing carbon monoxide dehydrogenase (to reduce CO2 to CO with 100% selectivity). The high efficiency of these enzymes on dye-TiO2 set a benchmark with unprecedented high turnover rates for solar H2 and CO generation reduction. Electrons for fuel generation must ultimately be provided from water and the Reisner group demonstrated recently that a hybrid system consisting of a cyanobacterial photosystem II (PSII) on a mesoporous indium–tin oxide electrode allows for highly efficient water oxidation to O2.[3] Photosystem II is Natures light-driven water oxidising enzyme and it sets a benchmark in terms of O2 evolution rate under ambient conditions for the development of synthetic catalysts.

The references below give a good introduction in the topic and the project. 

[1] E Reisner, D J Powell, C Cavazza, J C Fontecilla-Camps and F A Armstrong, J. Am. Chem. Soc., 2009, 131, 18457-18466

[2] T W Woolerton, S Sheard, E Reisner, E Pierce, S W Ragsdale and F A Armstrong, J. Am. Chem. Soc., 2010, 132, 2132-2133.

[3] M Kato, T. Cardona, A. W. Rutherford and E. Reisner, J. Am. Chem. Soc., 2012, 134, 8332-8335.

Learning outcomes and skills acquired: Our approach is highly interdisciplinary as we combine enzyme chemistry (the BBSRC student will work on this aspect) with synthetic (in the form of a synthetic photosensitiser) and materials chemistry. The BBSRC student must therefore be highly comfortable to work in a cross-disciplinary group and be interesting in thinking outside her/his formal field of training. We are also part of the Cambridge Algal Bioenergy Consortium will also be particularly relevant to this project.

Project availability: Michaelmas and Lent Term

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Growing algae at scale – use of microbial consortia to ensure robust and stable cultures

Supervisor: Prof. Alison Smith, Plant Sciences

Project abstract: Microalgae have emerged as a promising and sustainable biotechnological platform for the production of high value compounds, bulk and commodity chemicals, as well as biofuels. This is because they are photosynthetic, so can grow autonomously with relatively few inputs, many species grow fast, and the diversity of algae means that they synthesise a range of novel and useful compounds. Nevertheless, this is not yet a commercial reality for many reasons, not least the difficulty in growing sufficient algal biomass at scale throughout the year, and with the appropriate level of the product of interest. One of the major problems, particularly if the algae are grown in monoculture, is contamination by bacteria and other adventitious organisms. In the natural environment, algae live in consortia with other microbes, often exchanging nutrients in a mutualistic interaction. Over half of all microalgae require vitamin B12 (cobalamin) for growth, an organic micronutrient that is made only by bacteria. In our laboratory, we have established a number of model systems in which B12-dependent algae obtain the vitamin from heterotrophic bacteria in exchange for photosynthate. We study many aspects of these systems, including biochemical and physiological studies to determine the behavior of the different organisms, the molecular level to identify genes and proteins involved in facilitating the exchange of nutrients, and using systems approaches, including transcriptomics/proteomics and metabolic modelling to understand how the interaction is initiated and regulated. The information gained can then be used to inform the establishment of robust algal-bacterial consortia that will have both high productivity and resilience against contamination by other deleterious bacteria. The specific project will use proteomics data to identify genes that are upregulated in coculture versus axenic algal cultures, then verifying these by a combination of qPCR, western blot analysis, and biochemical analysis.

Learning outcomes and skills acquired: Analysis of proteomics data using proprietary software, querying of metabolic databases and pathway reconstruction; RNA extraction and qRT-PCR analysis; protein extraction, western blot and enzyme activity analysis.

Project availability: Michaelmas and Lent Term

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Extraction of medical collagens from food process waste

Supervisor: Professor Richard Farndale, Biochemistry

Project abstract:

Collagens are valuable materials in tissue repair and regenerative medicine, being highly conserved across species and intrinsically biocompatible.  They provide support to all organs within the vertebrate organism, and provide a substrate for cell attachment and migration.  Collagens are used in wound sealant and repair applications, as well as to form scaffolds in regenerative medicine.  Currently, collagens for medical use are extracted from bovine skin and cartilage as well as from fish skin.  The latter tend to be denatured, whilst the former must be free of transmissible disease, notably prions.  For this reason, bovine materials are usually sourced at substantial expense from Australia, considered a BSE-free zone.  In this project, a procedure will be developed to extract collagens from chick feet, usually discarded from the UK food industry.  The objective is to optimise a process that can be up-scaled from lab to pilot scale.  It will be picked up by local Biotech industry and developed further if successful.  The project will culminate in the testing of the product for cell-reactivity and in the preparation of collagen fibres from monomer suspensions.  There is a requirement for invention and problem-solving, along with physicochemical/biophysical skills.

Reference: Liu et al. 2001 Aust-Asian J Animal Sci 14 (11) 1638-44

Learning outcomes and skills acquired: The student will learn chemical and physical extraction technology; analytical procedures; materials handling skills.  During the testing phase, the student will learn cell culture and handling, and assays of cell-reactivity of the collagen preparations.

Project availability: Michaelmas and Lent Term

Other relevant themes: Basic bioscience underpinning health

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Exploring the accessibility of alternative mutational trajectories by directed evolution

Supervisor: Prof Florian Hollfelder

Project abstract: Identifying the constraints that shape the course of protein evolution is a long-standing question. More specifically, whether or not the evolutionary process is free to follow different trajectories during the acquisition and optimization of new protein functions still remains unclear1-3. We previously used directed evolution to investigate how a catalytically promiscuous arylsulfatase from Pseudomonas aeruginosa (PAS)4,5 can be converted into a phosphonate monoester hydrolase (PMH). Three rounds of neutral drift6, followed by six rounds of selection for improved target activity resulted in a highly generalist enzyme, displaying equivalent catalytic efficiencies for four chemically distinct substrates: phosphate diesters, phosphate, phosphonate, and sulfate monoesters7. Interestingly, the substitutions introduced during the evolutionary experiment clustered in loops that are absent in the structure of natural PMHs. This observation led us to design randomized deletion libraries targeted toward these regions, which enabled to identify variants tolerating up to 18 AA deletions and displaying compelling changes in specificity (>103-fold). The proposed project aims at addressing how different the evolutionary outcome would be upon the initial introduction of these function-altering deletions. Indeed, as such backbone mutations can induce significant structural changes that substitutions only cannot cause8, they may act as significant constraints to open alternative mutational trajectories in the conversion of PAS into a PMH. The planned work will consist of performing directed evolution experiments toward improved PMH activity using one of the previously identified deletion PAS variant as starting point. The techniques that will be used in this project include the generation of variant libraries using error-prone PCR and/or DNA shuffling, the screening of these libraries, production and purification of selected variants and their biophysical and biochemical characterization.

Project availability: Lent Term (January - March 2015)

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Convergent Directed evolution of phosphotriesterases

Supervisor: Prof Florian Hollfelder

Project abstract: Promiscuity refers to the ability of enzymes to catalyze other slow/small side activities besides their main native activity1. As such, this property is essential for the evolution of new enzymatic functions. Our aim is to evaluate the ability of different but similar enzymes to converge towards an increase of the same promiscuous activity using directed evolution. The comparison of different enzyme starting points and subsequent evolutionary trajectories would therefore mimic a convergent evolution process2. In Nature, the recent introduction of organophosphate pesticides (e.g. paraoxon) in the last century provided a new source of phosphorus for microorganisms, which rapidly acquired the ability of degrading such compounds by evolving phosphotriesterases (PTEs; e.g. paraoxonases)3,4. Recently, by screening environmental metagenomic libraries for paraoxonase activity, we isolated nine previously uncharacterized proteins. Kinetic studies subsequently revealed that these new enzymes were rather slow paraoxonases in comparison to well-characterized PTEs (kcat/KM ≈ 102 s-1.M-1 for our enzymes versus ≈ 108 s-1.M-1 for PTEs), suggesting that the metagenomic screen actually selected them by detecting one of their promiscuous activities. Interestingly, these enzymes are unrelated to any well-characterized PTEs known to date and belong to different protein families. Starting from these unrelated promiscuous paraoxonases, our convergent directed evolution approach will provide key informations on the structure-function relationships for the degradation of organophosphate pesticides. The proposed project will involve; the construction of enzyme variant libraries, the selection of variants improved for phosphotriesterase activity using an ultra high-throughput microfluidic technology developed in our lab5,6, the kinetic characterization of improved paraoxonase variants and the comparison of variants improved from different starting points in term of catalytic efficiency.

Project availability: Lent Term (January - March 2015)

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High-resolution drug discovery and high-throughput characterization at the picolitre scale

Supervisor: Prof Florian Hollfelder

Project abstract: This project will develop and use a new high-throughput screening platform of unprecedented capacity in which individual reactions are compartmentalized in water-in-oil droplets. The droplet is the equivalent of an ultra-small test tube. Droplet volumes are typically in the femto- to nanolitre range, which means that a large number of experiments can be carried out. The key idea is that the droplet compartment combines the functional molecule with all assay components that allow a readout of its function. When such droplets are generated in microfluidic devices, up to 10,000 highly monodisperse aqueous droplets can be generated per second (typically 10–200 µm in diameter corresponding to volumes between 0.5 pl and 4 nl) in a continuous oil phase.  Recently we have developed methods to generate over 1,000 data points time-courses to obtain high-resolution. We hope that this combination of high throughput and high quality will become a powerful tool in biology.  The goal of the project is to identify inhibitors of the enzyme dihydrofolate reductase (DHFR) from a small molecule library, to obtain potential anti-parasitic reagents. Assays for enzymatic activity will be miniaturized to the droplet scale and inhibition assays carried out on this ultra small scale. After improving the sensitivity of the assay to give reliable absorbance readings, kinetic screening in microtitre plates will be used to corroborate data obtained in droplets. The small molecules used for inhibition screens will be quantitatively evaluated and these data will be compared to hits obtained in a yeast screening assay (carried out in Steve Oliver’s Group).  This project involves collaboration with Bessie Bilsland in Steve Oliver’s group and possibly with Marko Hyvonen’s group (in a possible extension of this approach to fragment-based drug discovery). The project will includes technical developments of a novel high-throughput characterization tool based on encapsulation in micro-compartments. The novel platform will generate on-demand concentration gradients from minimal sample volumes in an automated fashion. To this end we will designing and set-up an optical interface (for fluorescence detection) to monitor large numbers (>104) of reactions in real-time, integrate it  with microfluidics, and develop software codes for the interface and post-processing of the data. A previously conducted directed evolution experiment on the specificity-switch from a sulfatase to a phosphatase will be used as a start enzyme collection from which mutants will be characterized. Promiscuity patterns will be quantitatively analyzed via measurement of kinetics parameters.

Project availability: Lent Term (January - March 2015)

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