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| − | <li><a style = "font-family: 'Raleway', sans-serif;" href="https://2018.igem.org/Team:Macquarie_Australia/Model">Our Model</a></li>
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| − | <li><a style = "font-family: 'Raleway', sans-serif;" href="https://2018.igem.org/Team:Macquarie_Australia/ModelExperimentation">Experimental Data</a></li>
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| − | <a style = "font-family: 'Raleway', sans-serif;" class="dropdown-toggle active">Collaboration<span class="caret"></span></a>
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| − | <li><a style = "font-family: 'Raleway', sans-serif;" href="https://2018.igem.org/Team:Macquarie_Australia/Collaboration">Overview</a></li>
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| − | <h style= "font-size: 25px; font-weight: bold;">Abstract</h>
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| − | 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, <i>Escherichia coli </i>. These vesicles allow for the sequestration of desired proteins and hence enable simplified, bulk purification via chromatography or centrifugation. Similarly, enzymes and small molecular natural products are typically cytotoxic to the cell may be sequestered. To address these issues, chlorophyll biosynthesis genes will be introduced into E. coli. These genes that have been identified as the source of prolamellar bodies (PLB) in photosynthetic organisms. Crucially, these PLBs become membrane bound compartments in once exposed to light. By recreating PLB formation in <i> E. coli </i> and exposing the cells to light, synthetic vesicles can be formed.
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| − | <h style= "font-size: 25px; font-weight: bold;">Approach</h>
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| − | It has long been known that E. coli produces protoporphyrin as a natural metabolite (Cox and Charles, 1973) 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 widely as a model organism. 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.
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| − | These cells, when grown in the absence of light, cause protochlorophyllide (pchlide) to accumulate into semi-crystalline aggregates with phospholipids and key enzymes. These aggregates, termed prolamellar bodies, are the foundation of our vesicles. The cells are subsequently exposed to light, activating the POR (protochlorophyllide oxidoreductase) enzyme, resulting in the production of chlorophyll 𝛼 from pchlide (Bradbeer et al. 1974). 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.
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| − | <h style= "font-size: 25px; font-weight: bold;">References</h>
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| − | 1.) Cox, R. and Charles, H.P., 1973. Porphyrin-accumulating mutants of Escherichia coli. <i>Journal of bacteriology</i>, 113(1), pp.122-132.
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| − | 2.) Bradbeer, J.W., Gyldenholm, A.O., Ireland, H.M.M., Smith, J.W., Rest, J. and Edge, H.J.W., 1974. Plastid development in primary leaves of Phaseolus vulgaris. <i>New phytologist</i>, 73(2), pp.271-279.
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