Difference between revisions of "Team:Macquarie Australia/Design"

The ultimate goal of our project was to produce vesicles in E. coli. We wanted to create a system that could enable simplified protein purification, as well as provide an isolated space for biochemistry within cells. During the first iteration of our design process, this general idea was our focus. After reviewing the literature, we realised that we could use the chlorophyll biosynthesis pathway to produce semi-crystalline aggregates of phospholipids and proteins called prolamellar bodies. When exposed to light, these spontaneously form vesicles. We saw this as a great opportunity to give chlorophyll an entirely new application within the biotechnology world. Ideally, our vesicles could be used in research labs, as well as on an industrial scale, in a broad range of projects. Before starting work in the lab, we needed to thoroughly design and plan our project, and to do this we established several guiding engineering principles.

• We wanted our vesicles to be simple to use. Multiple plasmids, unusual or complicated growing conditions, or an unusual expression vector were all to be avoided.

• To do this, we planned to assemble all of our genes onto a single plasmid with a single antibiotic resistance gene. This would enable any cell line to produce vesicles with a single transformation. Additionally, it would leave our cells useful for further transformations using other antibiotic resistance genes. Finally, we planned to optimise our project for use in E. coli. This is a ubiquitous cell with well established utility in industry and research, and an extensive catalogue of compatible synthetic biology parts available.

• We wanted our vesicles to be easy to isolate.

• To accomplish this, we envisioned bulk purification of target proteins or small molecules. We could capture compounds within our vesicles and separate them using centrifugation, or antibody affinity chromatography utilising membrane pound proteins within the vesicle membranes. Centrifugation was particularly desirable, as this would minimise the need for time and labor intensive chromatography steps in purification protocols.

• We wanted our vesicles to be easy to detect. Simple detection of vesicles would make purification of target compounds more efficient.

• In addressing this, we planned to use the unique spectral properties of chlorophyll to enable the simple and rapid spectroscopic identification of our vesicles.

• We wanted our vesicles to be useful and tunable as research tool and to comply with current synthetic biology standards.

• This involved packaging our final assembly of genes into a familiar biobrick layout, as well as providing sequences and samples of each of the simple parts. This would enable future researches to make adjustments as necessary.

• We had to be able to test for our vesicles, and make sure they were forming from the prolamellar bodies.

• We planned to observe our vesicles microscopically, using the naturally embedded chlorophyll as a UV reactive stain

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