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Developing and applying genetically encoded proteins as pre-resonant coherent Raman scattering tags for next-generation live-cell imaging


Project Description

EPSRC funded DTP PhD Studentship within the Interdisciplinary Doctoral Training Hub “Physics of Life”

The Hub is designed as a cross-disciplinary PhD research and training programme at the physics/life science interface. Students will benefit from joint supervision across the Colleges of Physical Sciences and Life Sciences at Cardiff University. Each project commences with two 3-months stages in the labs of the joint supervisors. The Hub will offer cohort development opportunities through joint research meetings, a “Physics of Life” summer school in 2022, and a student-led workshop in 2023. Students will be part of a vibrant, interactive community, sharing monthly newsletters and connected via a dedicated portal. Each student will be supported by a mentor. The Physics of Life Doctoral Training Hub aims to equip PhD candidates with multidisciplinary research skills that are highly sought after in academic and industry.

Your research project will develop next generation biological imaging methods by using synthetic biology to design and apply protein tags for Raman-based micro-spectroscopy. Optical microscopy is an indispensable tool that is pivotal to understanding biological processes in the cell and is currently the only practical means of obtaining high spatial and temporal resolution within living cells and tissues. Fluorescence microscopy has provided a highly sensitive and specific method of visualizing biomolecules and has revolutionised cell imaging, especially after the discovery of fluorescent proteins (FPs) that enabled genetic tagging of specific targets inside living systems. However, there are flaws, as fluorescent probes are prone to photo-bleaching and associated cytotoxicity which hamper their use, especially for imaging over long timescales in live cells which is critical to understanding biological process and underlying disease states. Moreover, the emission spectrum of these probes is quite broad, limiting simultaneous monitoring of multiple targets.

Your project will seek to merge the advantages of genetically encoded fluorescent proteins with those of vibrational Raman tags to develop a new live cell imaging approach. The underlying idea is to incorporate Raman-active chemical bonds not normally present in biology at specific locations near the functional centre of a FP. These new genetically encoded imaging probes will allow you to exploit a process called electronic pre-resonant Raman scattering (PRRS), enhancing the Raman signal by some 2-4 orders of magnitude, and therefore enabling imaging of a small number of these tags. Protein engineering together with a reprogrammed genetic code method will be used to incorporate non-natural amino acids containing Raman active chemical bonds at optimal positions in selected FPs, selected via in-silico design. Using advanced Raman scattering micro-spectroscopy you will quantify and understand PRRS in your designed proteins and apply them to image cellular events. Initially, you will generate tubulin-FP constructs to demonstrate the technique on static (microtubules) and dynamic (assembly/disassembly) cellular structures. Crucially, PRRS is expected to surpass traditional fluorescence approaches in terms of photo-stability, and you will verify this property by long term imaging throughout the cell cycle. This will in turn allow us to better understand the dynamics of tublin assembly/disassembly throughout the life time of a cell, and how anticancer and anti-inflammatory drugs impact on these processes.

The project lies at the physics/chemistry/life sciences interface and so will immerse you in an inter-disciplinary environment, a facet currently in demand in both academic and commercials sectors. The nature of the project means you be trained in a range of relevant techniques including genetic manipulation, protein design and engineering, cell biology, biophysics, advanced Raman micro-spectroscopy, quantitative analysis and coding.

You will also benefit from a supportive and experienced supervisory team that have established research groups with the required expertise and infrastructure. The supervisory team offers a combination of expertise at the physics/life science interface, with strong track records in engineering FPs using non-natural amino acids (Jones), developing novel optical microscopy techniques (Langbein/Borri), and imaging technologies applied to cell biology (Watson).

Funding Notes

This is a fully-funded studentship which includes fees, stipend and a research and training support grant, for 3.5 years at UKRI rate, for home/EU students.

Self-funded international students are also welcomed to apply

References

Wei, L. et al. 2017. Super-multiplex vibrational imaging. Nature 544, pp 465-470

Reddington, S. C.et al. 2015. Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence. Chemical Science 6, pp. 1159-1166.

Karuna A. et al. 2019 Label-free volumetric quantitative imaging of the human somatic cell division by hyperspectral coherent anti-Stokes Raman scattering, Anal. Chem. 91, pp. 2813-2821

How good is research at Cardiff University in Biological Sciences?

FTE Category A staff submitted: 54.70

Research output data provided by the Research Excellence Framework (REF)

Click here to see the results for all UK universities

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