Project Description

In the Beginning…

Over millennia, eukaryotic cells have evolved sophisticated organelles, which enabled them to partition their cytoplasmic contents into functional sectors (e.g. the nucleus for storage of genetic material). Such compartmentalisation allows greater efficiency of cellular processes, where each organelle is allocated a set of specific metabolic tasks. Some prokaryotes have also developed a method of forming intracellular subdivisions called bacterial microcompartments (BMCs) by expressing a set of proteins that ‘cage in’ a reaction pathway to make it more efficient. A wide range of metabolic pathways are isolated by BMCs, from carbon dioxide fixation pathways to organic compound degradation. Despite these varying functions, the proteinaceous BMC shells show considerable homology as a result of a highly conserved protein domains.[1]

One such set of proteins is expressed from the propanediol utilisation (Pdu) operon in Salmonella enterica, which is involved in the coenzyme B(12)-dependent breakdown of the organic compound 1,2-propanediol. S. enterica synthesises its microcompartments during aerobic or anaerobic growth on 1,2-propanediol, but does not form them when grown on other carbon sources. Enzymes vital for the catabolic pathway are thought to be enclosed within the BMC.[2]

Figure 1: Gene organization in the Pdu Operon and Pdu metabolic pathway[1]

The Pdu operon is composed of 21 genes, which encode proteins involved in the Pdu microcompartment (Figure 1) either in formation of the shell or in the metabolic pathway contained inside. Of these 21 genes, seven have been identified as having a role in BMC shell assembly. These genes code for the proteins PduA, -B, -J, -K, -N, -T, and -U plus a truncated version of PduB termed PduB’.[3]

It is thought the Pdu BMC shell adopts an icosahedral shape, with multiple flat triangular faces and 12 vertices. With the exception of PduN, the above Pdu shell proteins form a mosaic of hexagonal building blocks that associate into flat sheets to form the facets of the icosahedron. While the single-BMC-domain proteins PduA, -J, -K, and -U (Figure 2) form protein hexamers, PduB, -B’, and -T have tandem BMC domains so instead form hexagonal protein trimers.[4]

Figure 2: Crystal Structure of the PduU Shell Protein5

Small pores, approximately 10Å in diameter, run through the centre of some hexagonal units and are thought to allow transport of molecules across the protein shell3. PduN forms pentamers that occupy the 12 vertices of the icosahedron and is therefore considerably less abundant than the other Pdu shell proteins which contribute hundreds of copies to the faces of the BMC[4].

Figure 3: Icosahedral Bacterial Microcompartment[6]

New Biobricks and Improving Old Biobricks

The Grand Plan: A Synthetic Microcompartment

To begin with, our team aims to produce a BMC comprised of the eight Salmonella Pdu shell proteins (Pdu-A, -B, -B’, -J, -K, -N, -T, and -U) in Escherichia coli, which itself is a close relative of Salmonella. This involves expressing seven Pdu genes, as PduB’ is expressed from the PduB gene sequence. Our intention is to add a poly-histidine affinity tag onto the C-terminus of several compartment proteins, which will aid in determining whether the proteins have been expressed and may allow us to isolate complete compartments from the cell by affinity purification.

In order to achieve expression of the BMC in E. coli, we will construct a biobrick that contains all seven genes for the Pdu shell proteins, including sequences encoding the poly-histidine affinity tags. In order to create our biobrick, we will employ a progressive strategy involving construction of individual Pdu gene biobricks and sequential addition of these biobricks to form our complete synthetic operon (Figure 4). As the majority of the Pdu shell genes lie adjacent to one other shell gene in the Pdu operon of Salmonella, it is possible to clone these genes as pairs. Therefore our initial individual biobricks will consist of PduAB, PduJK, PduN and PduTU.