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Developing ultra-small protein visualization tools using synthetic biology and organic chemistry

Primary Supervisor: Professor Mohan Balasubramanian, Warwick Medical School

Secondary supervisor: Mike Shipman, Chemistry

PhD project title: Developing ultra-small protein visualization tools using synthetic biology and organic chemistry.

University of Registration: University of Warwick

Project outline:

A key goal in cell biology is to describe the intracellular distribution and dynamics of all proteins within the proteome in normal and diseased states. Although fusing proteins with fluorophores (or fluorochrome binding-peptides) is extremely powerful and has revolutionised imaging intracellular protein dynamics, in many cases the large tag appended to proteins also compromise their function. This issue is particularly true for major cytoskeletal proteins (e.g. actin and tubulin) and micropeptides, which cannot be labelled in a functional form.

To address this limitation, we will radically improve the ability to investigate intracellular proteins by incorporating a ~0.4 kDa fluorescent amino-acid (derived from fish skin Bromo-kynurenine ‘B-kyn’: excitation-375nm and emission-508nm), which is ~ 1/80th the size of the commonly used green fluorescent protein, directly into target proteins using a combination of organic chemistry and synthetic biology.

We have already developed a synthetic pathway to make B-kyn. The student will establish methods to create and test derivatives that can be conjugated to proteins in a variety of ways using different chemistries (e.g. azido, maleimide) or by modification of different amino acids (e.g.lysine, phenylalanine, tryptophan). The student will also develop red-shifted fluorescent amino-acids with enhanced properties, which will be used to generate fluorescent proteins. The student will develop expertise in organic chemistry approaches and will be able to design and synthesize compounds with interesting biological properties. This part of the work will be carried out in the laboratory of Mike Shipman.

The student will evaluate the effectiveness of B-kyn incorporation into proteins within bacteria, yeast, and mammalian cells using genetic code expansion technology and biochemistry and investigate the function in in vitro and in vivo studies. In this section, the student will develop synthetic biology, biochemistry, and cell biology skills. The student will use orthogonal pairs of tRNAs and tRNA synthetases to incorporate using the normal translation mechanism, B-Kyn into proteins within cells. B-Kyn-bearing proteins and cells will be characterized using the methods of biochemistry and advanced imaging. This part will be carried out in the group of Mohan Balasubramanian.

The work the student performs can potentially be transformational in the study of protein dynamics in vitro and in vivo. The student project also has potential to lead to patent development and industry partnerships.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology, Microbiology, Neuroscience and behaviour, Plant Science, Stem Cells, Structural Biology, Systems Biology

­Techniques that will be undertaken during the project:

    • Organic Synthesis
    • Genetic Code Expansion
    • Advanced Imaging
    • Biochemistry, Protein Purification
    • Bacterial, yeast, and mammalian biology

    Contact: Professor Mohan Balasubramanian, University of Warwick