Identifying NMDA receptor complex interactomes of epilepsy associated genetic variants using BioID( Ref: WD_2022_21_WSCH)

Post Summary

Applications are invited from suitably qualified candidates interested in pursuing a PhD in the area of molecular biology on a recently funded research project within the PMBRC at WIT. This project will involve molecular biology, mammalian cell culture, mass spectrometry and BioID based proteomics.

Epilepsy is a debilitating neurological disorder that affects approximately 1% of the global population. One of its main causes are genetic mutations that result in ion channels behaving abnormally. However in many cases we don’t know what makes up these ion channel complexes, or how these mutations lead to the development of epilepsy. We are interested in discovering which proteins are present in both healthy cells and in cells that have epilepsy linked genetic variations, in order to understand which molecules are critical in the development of epilepsy.

The central aim of the project is to identify NMDA receptor interactomes using BioID. BioID involves fusing a modified promiscuous biotin ligase enzyme, BirA*, to a bait protein. When the fusion protein is expressed in a cell it biotinylates proteins that interact with the bait. The interacting proteins can then be isolated and subsequently identified by mass spectrometry.

It is well established that genetic variations such as single nucleotide polymorphisms (SNPs) are associated with disease acquisition. However, typically very little is known about which molecules are involved in disease progression. The main objective of this project is to identify protein interactomes for both “wild-type” and known epilepsy associated variants of NMDA receptors (NMDARs) using BioID. Identifying the differences in protein interactomes between these cohorts using Differential Interactome Analysis (DIA) will enable us to build a profile of proteins and signalling networks that may contribute to the progression of epilepsy.

 Specific Research Objectives

Objective 1 – To determine the protein interactome for NMDA receptors in cell culture using BioID.

Objective 2 – To determine the protein interactomes for a range of epilepsy linked NMDA receptor variants in cell culture using BioID.

Objective 3 – To identify, characterise and validate signalling differences between wild-type and epilepsy linked NMDAR variants, establishing molecular pathways for progression of the disease. 

 This project follows the WIT structured PhD model, which means the successful PhD student will complete 30 credits of taught modules (Universal path modules and discipline specific modules related to the project). In addition, hands on training in cell culture, molecular biology, mass spectrometry and BioID will be provided as required.

Person specification

Qualifications

Essential

·      Applicants should hold or expect to attain, at a minimum a 2:1 Honours Degree or equivalent in Molecular Biology, Biochemistry, Cell Biology, Biopharmaceutical Science, Pharmaceutical Science, or related area.

 Desirable

·      1^st class Honours Degree

·      Knowledge of molecular biology, cell culture, mass spectrometry or proteomics as part of their undergraduate degree or work experience.

Knowledge & Experience

Essential

·       A research project carried out in one of the above disciplines as well as having an associated thesis written.

Desirable

·       Work placement undertaken in an industry related to the above disciplines.

·       A demonstrated knowledge of a number of the following: mammalian cell culture, mass spectrometry, proteomics, protein purification, bioinformatics (especially analysis of SNPs and proteins), molecular biology techniques either DNA based (eg. PCR, site directed mutagenesis, agarose gels), or protein based (eg. Western blots, Co-Immunoprecipitation).

Skills & Competencies

Essential

·       The applicant must have a high level of self-motivation, be able to think and work independently.

·       Good communication skills. Applicants must be able to present their work orally at meetings and at conferences.

·       Good scientific writing skills. Applicants must be able to communicate their work to the general public and via scientific literature.

·       Good interpersonal skills and a demonstrated ability to work in a collaborative environment.

·       High level of computer literacy.

·       Applicants whose first language is not English must submit evidence of competency in English, please see WIT’s English Language Requirements for details.

Desirable

·       Knowledge of scientific database searching and reference management software. 

·       Evidence of interest in the topic under study.