Team:Macquarie Australia/Description

Abstract

poteins need to be synthesised and purified for use in therapeutic and industrial applications. At present, this process is costly, time-consuming and operationally difficult. We aim to address these challenges through the formation of vesicles within a familiar and ubiquitous expression vector, Escherichia coli. These vesicles allow for the sequestration of desired proteins and hence enable simplified, bulk purification via chromatography or centrifugation. Similarly, enzymes and small molecules also present the opportunity to produce or process natural products and refine typically cytotoxic compounds. To address these issues, the chlorophyll biosynthesis genes will be introduced into E. coli, and it is these genes that have been identified as the source of prolamellar bodies (PLB) in photosynthetic organisms. The formation of vesicles within E. coli is novel research, never attempted in prokaryotes before and would act as a toolkit to address the multitude of issues mentioned above.

Hypothesis

Expression of the chlorophyll biosynthesis pathway in E. coli will lead to PLB formation, when grown in the dark. When exposed to light, the PLB will dissociate and leave behind the lipid structure.

Approach

Previous research has demonstrated that E. coli produces protoporphyrin as a natural metabolite and we will be using this as a starting point for chlorophyll biosynthesis in E. coli. To do this, we will be using the genes from the chlorophyll biosynthesis pathway in Chlamydomonas reinhardtii, a well studied alga used as a model in laboratories. In support of this, we have carried out literature searches to identify the expression level necessary for each gene and optimised this through computer modelling. Based on this research, several operons have been designed, each with the optimal expression levels necessary. Once complete, each of these operons will be assembled into one plasmid designed around a standardised biobrick design. This allows for vesicle formation to be introduced into any E. coli culture via a single transformation.
These cells, when grown in the absence of light, cause protochlorophyllide (pchlide) to accumulate into semi-crystalline aggregates with phospholipids and key enzymes, forming PLBs, the foundation of our vesicles. The cells are subsequently exposed to light, activating the POR (protochlorophyllide oxide reductase) enzyme, resulting in the production of chlorophyll 𝛼 from pchlide. This conversion has been experimentally demonstrated to result in the spontaneous formation of phospholipid vesicles from the PLBs. Thus, we will enable E. coli to produce vesicles, generating a tool that can be used in research and industry with profound implications.