EASTBIO Investigating the Assembly of Bacterial Encapsulins for the Efficient Engineering of Nanoscale Bioreactors

Project Details:

Encapsulins are bacterial capsid-like nanocompartments roughly 25-30 nm in diameter. These large protein assemblies are made up of either 60 or 180 copies of a single 30 kDa protomers which assemble to form a hollow icosahedral protein cage. A partner enzyme is sequestered inside the encapsulin by an interaction between a short encapsulation sequence (ES) appended to the protein and the interior of the encapsulin shell. The resulting complex offers a privileged environment for performing catalysis and a store for potentially harmful products or intermediates in enzymatic pathways (1). 

Several different types of enzymes have been identified as encapsulin cargo-proteins – including peroxidases and ferritins. However, recent work has also demonstrated that the ES sequence can be grafted onto heterologous proteins to localise them to the lumen of the encapsulin, and several heterologous reporter proteins have been directed to encapsulins in this way. This ability to encapsulate non-native proteins, coupled with the potential to engineer the outer-surface and pores of the encapsulin shell, affords these systems great potential as nano-machines for biocatalysis, bioremediation, and as therapeutic tools (2).

 Little is currently known about the loading stoichiometry of native or heterologous cargo proteins within encapsulins; how these complex systems assemble in vivo; or how loading stoichiometry effects the enzyme activity and the storage capacity of these systems. Understanding the structure, biochemistry and biophysics of these systems is required to fully realise their potential as powerful nanotechnology and synthetic biology platforms.

 In this PhD project the student will produce a series of native and heterologous encapsulated systems using an established modular synthetic biology platform. In collaboration with the Wallace Lab, a series of industrial biocatalytic target enzymes will be chosen for encapsulation and the effect of encapsulation on stability and activity will be determined. The assembly of these encapsulated systems will then be monitored using a combination of several structural mass spectrometry approaches. Native mass spectrometry and ion mobility mass spectrometry techniques allow the observation and characterisation of intact encapsulin systems (3). By combining this technology with rapid mixing techniques, developed in the Clarke Lab (4), we will observe encapsulin stepwise assembly. These studies will outline the parameters for efficient encapsulation of proteins in these systems and providing a framework to produce designed encapsulated enzymes.

The student will be trained in synthetic biology techniques, protein production, purification and biochemistry. In addition, the student will gain hands-on experience in structural mass spectrometry and will develop new structural mass spectrometry techniques. Experiments will be performed on the BBSRC-funded state-of-the-art high field Fourier transforms ion cyclotron resonance mass spectrometer (FT-ICR MS) housed at the School of Chemistry.

Application Process:

To apply for an EASTBIO PhD studentship, follow the instructions below:

·        Informal enquiries should be addressed to Dr David Clarke. To apply, please send a cover letter outlining your previous research experience and reasons for applying, alongside an up-to-date CV to [Email Address Removed]

·        After you have discussed the projects of interest to you with Dr David Clarke, download and complete our Equality, Diversity and Inclusion survey and then fill in the EASTBIO Application Form and send this to Dr Clarke.

·        Send the EASTBIO Reference Form to your two academic/professional referees and ask your referees to submit your references directly to Dr David Clarke [Email Address Removed]

We anticipate that our first set of interviews will be held 6^th – 10^th February 2023.

If you have further queries about the application/recruitment process please contact EASTBIO

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