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
With engineering principles established, we were able to begin identifying the genes we would need to construct our plasmid. We chose to use genes from Chlamydomonas reinhardtii, as considerable research already exists on its chlorophyll biosynthesis pathway. Additionally, we were able to use some of the parts previously assembled by former Macquarie teams.
Our plasmid was divided into three functional portions. The first was a series of genes that formed the magnesium chelatase complex. These genes encode proteins that function to insert a magnesium ion into the porphyrin ring of protoporphyrin, a natural metabolite within E. coli. This would produce Mg-protophorphyrin IX. The genes required for this portion of the plasmid were:
- ChlH
- GUN4
- ChlI1
- ChlID
- ChlI2
Next, we identified a series of genes that would be necessary convert Mg-protophorphyrin IX into protochlorophyllide. This made up the second portion of our plasmid. These genes were:
- ChlM
- CTH1
- Ycf54
Following this, we needed a series of genes would would enable the conversion of Mg-protophorphyrin IX into chlorophyll. This was the third and final portion of our plasmid. These genes were:
- POR
- ChIP
- DVR1
- ChIG
In addition to chlorophyll biosynthesis genes, we planned to incorporate the ycf39 gene. This was based on literature reports which indicated this protein may be necessary for the formation of prolamellar bodies.
KARL PLEASE MAKE ME A NICE PICTURE
Before starting work in the lab, we looked at the work of previous Macquarie teams and the plasmids they had partially constructed. We computationally modelled chlorophyll production and realised we could improve the efficiency of this process by increasing the expression of several genes. In particular, increasing transcription of the ChlI1, POR, fdx and FNR genes improved the efficiency of chlorophyl production. As such, we decided not to use many of the parts assembled by previous Macquarie teams. We created the second iteration of our design, in which a trc protomoter was placed before the aforementioned genes. Modelling this promoter swap based on gene expression data obtained by Tegel, Ottosson, and Hober (2011), we were confident our improved plasmid would be more efficient at producing chlorophyll.
