Biochemistry and Biophysics PhD research projects

Supervisor: Bruce, Professor Neil C

Mining composting communities for new lignocellulose mobilising enzymes (2015-16)

From both a fundamental and industrial biotech viewpoint understanding the deconstruction of lignocellulose in soil and compost is of central importance. In the natural environments microbial communities can efficiently degrade or modify lignin to enable the effective enzymatic hydrolysis of the polysaccharides present in plant cell walls. Globally, this is important for cycling carbon in the environment and as potential sources of biocatalysts for efforts at converting plant biomass into biofuels and commodity chemicals. The objectives of this project are to use metatranscriptomics and proteomics to determine gene- and protein-centred details to determine new mechanisms and improved methods of lignocellulose deconstruction in mixed microbial communities from composting cereal straw. The project will use proteomics analysis to interrogate the secretome of microbial communities in composting cereal straw and metatranscriptomics will be used to explore the expression of genes associated with lignocellulose digestion. To identify new linocellulose degrading enzymes, the peptide sequences from the proteomics analysis will be used to probe the metatranscriptomic library for full and partial coding sequences. These coding sequences will be cloned, expressed and the recombinant proteins characterised.

Co-directors:Simon McQueen-Mason

Supervisor: Chong, Dr James

Please email to discuss potential projects

Supervisor: Baumann, Dr Christoph

Design and characterisation of novel fluorescent nucleotide substrates for RNA polymerases (2015-16)

The ability to follow the polymerisation of ribonucleotide triphosphates (NTP) by a DNA-dependent RNA polymerase using fluorescence techniques requires NTP analogues with a fluorophore attached to either the gamma phosphate or the nucleobase. Attachment of a fluorophore at these positions allows both base pairing with the template DNA and phosphodiester bond formation. NTP analogues with the fluorescent group attached to the nucleobase have the added benefit that the newly synthesised polynucleotide chain is rendered fluorescent. In collaboration with the Department of Chemistry at York, we are developing novel classes of fluorescent NTP analogues. This project will involve studying the photo-physical properties of these analogues using ensemble-averaged and single-molecule fluorescence spectroscopy techniques, and following their incorporation into nascent RNA chains by single- and multi-subunit RNAPs using equilibrium and kinetic methods.

Co directors - Ian Fairlamb

Supervisor: Ungar, Dr Daniel

Connecting vesicle targeting with SNARE complex assembly (2015-16)

The 2013 Nobel prize in Medicine was awarded for the discovery of the protein factors involved in intracellular vesicle transport. Vesicle transport is critical for processes such as neurotransmitter release or insulin regulated GLUT4 secretion, and therefore impacts on areas like neuroscience and diabetes. Yet the regulation of the final step of vesicle trafficking, the fusion of the two membranes is not well understood. It is well known that a SNARE complex that bridges the two membranes has to form to fuse these membranes, but how SNARE complex formation is regulated is an important open question. We would like to understand how the process of vesicle targeting regulates SNARE complex formation and thereby membrane fusion. During vesicle targeting the first contact between the vesicle and target membrane are made, and therefore this is a key site for vesicle trafficking specificity – where does a vesicle end up in the cell. Large multisubunit tethering complexes (MTCs) coordinate vesicle targeting throughout the cell, one working at the Golgi apparatus is the COG complex. COG is important for the sorting of Golgi-resident proteins, most notably glycosylation enzymes, but also Golgi SNAREs. COG is known to interact with several Golgi SNAREs, yet if this interaction is involved in regulating SNARE complex formation is not established. We will use biochemical SNARE assembly assays with purified COG to address this question. Mutant COG complexes will also be generated, which cannot bind SNAREs, and tested both in the biochemical assay and for their ability to alter sorting within the Golgi. Golgi sorting will be assessed by analyzing the glycosylation potential for cells using a mass spectrometric glycan profiling method. Thus this project will combine biochemical, cell biological, glycobiological and mass spectrometric methods for the analysis of an important cell biological problem.

Miller VJ, Ungar D (2012) Re’COG’nition at the Golgi. Traffic, 13, 891-897

Co directors - Nia Bryant

Supervisor: Mark, Professor Leake

Single-molecule biophysics (2015-16)

A variety of PhD projects are available to enthusiastic, competitive self-funded graduate students in a world-class lab in the general area of single-molecule biophysics, involving utilizing either in vivo single-molecule fluorescence imaging techniques, or in vitro single-molecule manipulation and superresolution imaging using magneto-optical tweezers combined with superresolution microscopy, or a combination of both. Biological systems under study currently include DNA topology control, bacterial DNA replication, bacterial DNA repair, bacterial cell division, and yeast signal transduction. Projects could include elements of practical molecular biology, DNA construct design and protein purification, chemical conjugation technologies, application of single-molecule biophysics technologies and, if appropriate to the student, design and construction of new single-molecule biophysics technologies

  

Developing a single-molecule ‘Enviroscope’ to probe molecular processes in living cells in real-time. 

Biological processes in living cells possess enormous complexity involving highly cooperative and coordinated effects between remarkable, nanoscopic molecular machines composed of individual protein molecule components. The PhD student will work with Prof. Leake in the Depts of Physics and Biology at York. Prof Leake is the Chair of Biological Physics and the Director of the Biological Physical Sciences Institute (BPSI) at York encompassing multiple exceptional research teams across several different departments of the University, including Physics, Biology, Chemistry, Electronics, Mathematics, Psychology and Computer Science, which serves to nurture collaborative research at the cutting-edge interface between the physical and life sciences (http://york.ac.uk/physics/bpsi ). Prof. Leake’s team has an international reputation in the fields of single-molecule biophysics. The University of York is an exciting, young institution with a strong focus on cutting-edge interdisciplinary research.

The project will apply new bespoke/home-built super-resolution optical microscopy methodologies to image multiple protein components simultaneously in single, living cells down to a precision of single molecules with a spatial precision of a few tens of nanometre and a millisecond temporal resolution, permitting the real-time observation of functional molecular machines across all spatial regions of single live cells [1, 2].  The project will involve the design and construction of a new single-molecule ‘Enviroscope’ light microscope  – a device which can tracking single fluorescently tagged molecules in addition to quantifying  their spectral emission profile as a function of emission wavelength. This spectral emission property is a signature for the local physical and chemical environment, and so can be used to probe local pH, ion concentrations, hydrophobicity, viscosity and many more important physical parameters in the living cell. The student will work with an expert team to learn aspects of optical design, in addition to cellular growth and preparation and several invaluable cell biology and biochemistry skills. The new device will be applied to a range of biology questions in live cell studies through several current collaborations in the Leake lab, including DNA replication, gene regulation and signal transduction.

It is a unique interdisciplinary opportunity for a highly motivated student to engage at the exciting interface between the life and physical sciences and gain an exceptional PhD education in cutting-edge science.

For more information please contact mark.leake@york.ac.uk

  

Combining magnetic and optical tweezers with super-resolution imaging of DNA to probe new candidate antibiotics. 

We are seeking a talented and ambitious PhD student who can push forward developments of the core technologies of a prototype diagnostic system for bacterial resistance to antibiotics which utilizes molecular biosensing of the key antibiotic target enzyme DNA gyrase. The device uses a novel combination of mechanical/optical detection from single-molecule pull-down binding to synthetic DNA target constructs.  The PhD student will work with Prof. Leake in the Depts of Physics and Biology at York. Prof Leake is the Chair of Biological Physics and the Director of the Biological Physical Sciences Institute (BPSI) at York encompassing multiple exceptional research teams across several different departments of the University, including Physics, Biology, Chemistry, Electronics, Mathematics, Psychology and Computer Science, which serves to nurture collaborative research at the cutting-edge interface between the physical and life sciences (http://york.ac.uk/physics/bpsi ). Prof. Leake’s team has an international reputation in the fields of single-molecule biophysics. The University of York is an exciting, young institution with a strong focus on cutting-edge interdisciplinary research.

The project will use and further adapt a powerful new technology developed in the Leake lab which combines magnetic and optical tweezers to tether, extended and twist single molecules and DNA [1] whilst simultaneously imaging them using super-resolution fluorescence microscopy [2].  The objective of the project is to develop a prototype diagnostic device which uses the mechanical responses of specifically designed linear DNA probes to the binding of DNA gyrase from lysed E. coli cells and the gyrase-targeting antibiotic ciprofloxacin. This will be used to to determine if gyrase is resistant to the action of ciprofloxacin in addition to probing novel variants of ciprofloxacin developed by Prof. Leake’s collaborators in the Chemistry Dept. The student will work with an expert team to learn aspects of experimental single-molecule biophysics, in addition to computational and mathematical analysis tools, biochemical preparation procedures and chemical synthesis  techniques. The ultimate aim is to miniaturize this new device to generate a handheld machine which can be used in clinics.

It is a unique interdisciplinary opportunity for a highly motivated student to engage at the exciting interface between the life and physical sciences and gain an exceptional PhD education in cutting-edge science.

For more information please contact mark.leake@york.ac.uk

 

Supervisor: Maathuis, Dr Frans

Plant Arsenic Biomonitors (2015-16)

Arsenic (As) is a Class 1 carcinogen with no minimum threshold intake. Arsenic poisoning affects more than 100 million people worldwide, particularly in south Asia. Recent studies have identified foods, especially rice, as an important source of inorganic As intake by humans posing a potentially significant health risk for people on a rice-based diet living anywhere in the world1,2). The main reason for this phenomenon is the much greater efficiency with which rice accumulates As, compared to other cereal crops. In the worst affected areas, build-up of As in soil has also resulted in significant yield losses due to As toxicity. Since rice is a staple food for about half of the world population, there is an urgent need to develop strategies to limit As contamination, and an early warning system to signal the onset of As accumulation would be highly beneficial.

Objectives: We will generate crop plants that to report on their As status. To accomplish this we will use an arsenic sensor and a reporter. For the sensor, we will make use of the promoter of a gene that responds to As, the cytochrome P450 (Os01g43710) is an excellent candidate, and link this to a gene that when expressed provides an easily distinguished colour marker as reporter. For the latter, anthocyanins (red-purple pigments) are ideal. This approach will result in plants that will turn red whenever As toxicity presents itself.

Co director: Michael Schultze

Improving Arsenic tolerance in Rice (2015-16)

Project and background:  Arsenic (As) is a metalloid, which occurs ubiquitously in nature. It is found predominantly as inorganic arsenate and arsenite. Arsenate, as a phosphate analogue, has detrimental effects on phosphate metabolism but arsenite is even more toxic due to its high reactivity with sulfhydryl groups of proteins. As such As is classified as a group 1 carcinogen and there is large concern about human contact with As which occurs through drinking of contaminated water and via the food chain because in many areas crop growth depends on usage of As contaminated irrigation water. It is estimated that more than 100 million people are exposed to toxic levels of As.

Arsenic contamination through consumption of crops is particularly prevalent where rice is concerned: Rice is the staple of billions, it is produced in many south east Asian countries that have high levels of As in their aquifers, and rice translocates a relatively high proportion of As to its edible part, the grain.

No specific As efflux mechanism has been identified in plants. You will use a GWAS (genome wide association studies) approach to identify As efflux mechanisms. To assess if As efflux in plants can be augmented, and whether this has positive effects on plant growth, you will express heterologous systems such as ACR3 from Saccharomyces cerevisiae and/or use transgenic approaches (e.g. overexpression, genome editing) to alter activity of putative As efflux mechanisms in rice. Transgenic rice will be evaluated for As tolerance and As content level in various tissues such as roots, leaves and in seeds. This project will train the candidate in whole plant physiology, molecular biology and crop biotechnologies such as cereal transformation.

Improving Rice Salt and Drought Tolerance (2015-16)

Background: Salinity and drought stress are a major and global detriment to agricultural production. Their negative impact on crop production is exacerbated by sensitivity of major crops such as wheat and rice, a growing human population and climate change. For both salt and drought stress, the manipulation at the transcript level of single transporter genes in rice has shown that its tolerance can be significantly improved in this way. This includes altering expression of K+ transporters such as TPKs and AKTs and Na+ transporters such as NHXs and HKTs. However, the multigenic nature of salt and drought stress means that the expression of multiple transporters needs altering to optimise stress resistance. Such ‘stacking’ or ‘pyramiding’ of traits has hardly been attempted in rice abiotic stress.

Work plan and aims: This project envisages combining the positive effects that were observed by the manipulation of single genes to further improve salt and drought tolerance in rice. We will focus initially on genes for which transgenics are already available in the lab (vacuolar channels TPKa and TPKb, the K+ uptake and translocation systems AKT1 and SKOR and the Na+ transporter HKT2;1). Lines will be crossed and/or retransformed to generate multiple transgenics. Retransformation may include the use of tissue specific promoters. State of the art genome editing will also be applied to alter activity of specific genes. Using growth assays and mineral content analysis, lines will be assessed for synergistic phenotypes regarding tolerance to salt and drought stress.