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Research Experience

MIBTP Mini-Projects and Professional Internship for PhD Students

Professional Internship for PhD Students (PIPS)

Community and Education Development Officer: Cawthron Institute – New Zealand (July – September 2015)

The Cawthron Institute was a unique opportunity to gain insight into the importance of science communication and education within the wider community. Skills required for communicating science to the general public as well as specialist audiences were developed and refined. An interview at a local radio station and the videoing of my scientific presentation on “The antimibiotic apocalypse” were two unique opportunities I was able to exploit to analyse and reflect on and enhance my presentation technique. A Knowledge of programming in BASIC was used to create a versatile programme for the Community and Education Department to assist in the allocation of students, and judges at a large annual Science Fair in New Zealand with a variety of automated outputs including timetables and acceptance letters.

Mini-Project 2

Kinetic investigations into substrate analogues of S.pneumoniae and P.aeruginosa MurF as potential antimicrobials: School of Life Sciences, University of Warwick (April – June 2015)

Supervisors: Dr David Roper and Dr Adrian Lloyd

A structure activity relationship study, aimed at developing a ‘torjan-horse’ antimicrobial compound, towards MurF was conducted. MurF was expressed and purified using a combination of IMAC, SEC and ion exchange chromatography. The Michaelis-Menton kinetics of the natural substrate and five structural substrate analogues was determined using simple coupled spectrophotometric assays. All analogues were suitable substrates for the MurF enzyme, however the catalytic efficiency was considerably lower that that of the natural substrate. Crystallization trials were conducted, aimed at obtaining structural information about MurF in complex with the substrate analogues for iterative structural analogue design.

Mini-Project 1

Purification and Crystal Screening of MglABd-Bd2492, Bd2492 and RomR mutants: School of Biosciences, University of Birmingham (January – March 2015)

Supervisor: Dr Andrew Lovering

Structural characterisation of proteins believed to be involved in control of predation in the deltaproteobacteria Bdellovibrio bacteriovorus. A combination of molecular biology techniques including cloning and PCR were used to generate mutant protein constructs. Proteins were overexpressed and purified using a combination of Immobillised Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC). Crystallisation trials were conducted on the MglABd-Bd2592 complex as well as RomR and Bd2492 individually, crystallisation conditions were subsequently optimised, however, diffraction-quality crystals were not obtained.

Other Research Projects

Undergratuate Dissertation

The use of fluorescently-labelled single cysteine mutants in investigating interactions between auxilin and its binding partners, clathrin and Hsc70: School of Life Sciences, University of Warwick (2014)

Supervisor: Dr Corinne Smith

An investigation into the interactions between auxillin and its binding partners, clathrin and Hsc70, using fluorescently labelled single cysteine mutants was conducted. Real-time perpendicular light scattering was used to demonstrate that fluorescently labelled mutants of clathrin and Hsc70 did not affect disassembly of clathrin cages. In a novel study, the potential for use of time-resolved fluorecence anisotropy in understanding clathrin cage dissassembly was investigated. The proof of principal study found that time-resolved fluorescence anisotrpic changes were consistent with current understanding of the system and could hold unexplored promise and potential for understanding disassembly at a molecular level.

Intercalated Year Industrial Placement Studentship

Generation of a panel of anti-L-myc monoclonal antibodies: Tool mAb Group, Glaxo Smith Kline, Stevenage (2012 – 2013)

Generating a panel of tool anti-L-MYC monocolonal antibodies (mAbs) for a GSK research group. Immunogenic peptides were designed bioinformatically and created for immunisation protocols with SJL/OlaHsd mice. Techniques required for mAb generatopm included preparation of splenocytes, electrofusion for hybridoma generation and aseptic mammalian tissue culture. Hybridoma colonies were screening using ClonePix2 and a panel mAbs were purified using AKTA. mAbs were validated for use in a number of standard laboratory tecniques including ELISA, Western blot and Flow Cytometry. The final panel of mAbs were validated for use in ELISA and Western blot and showed specific binding to all three isoforms of the L-MYC protein with no cross-reactivity to C- or N-MYC proteins.

Undergraduate Research Support Scheme (URSS)

Reconstruction and Corruption of Streptococcal Peptidoglycan synthesis - Substrate analogues and inhibitors of Streptococcus pneumoniae MurE as potential antimicrobials: School of Life Sciences, University of Warwick (2011)

Supervisor: Adrian Lloyd

Structural analogues of the natural substrate (lysine) for MurE, were kinetically characterised using simple coupled spectrophotometric assays. Four sequential steps (MurC – F) in peptidoglycan precursor synthesis were recapitulated in vitro using two spectrophotometric assays. Five out of ten analogues investigated were shown to be substrates of MurE, but were less than 1% as efficient as the natural substrate, a further two analogues were identified as MurE inhibitors. Each of the successful substrate analogues generate product that could be processed by MurF indicating that ‘tojan-horse’ targeting holds promise for the future.