Team:UChicago/Design
Design
Our proposed system involves the intricate interplay between the 3 plasmids: the FASyn operon, the Alk operon and the Exporter operon (see parts page). The FASyn operon contains all of the relevant genes for fatty acid synthesis (FAS) including acyl-carrier protein. The Alk operon contains all constituent parts from the part Bba_K325909 as well as our new gene for the enzyme fatty-acid-photodecarboxylase which converts fatty acids into alkanes.
During the day, white light from the sun inactivates the photosystem AII (psbAII) promoter and thereby the transcription of the Fatty-Acid-Decarboxylase (FAP) which converts long chain free fatty acids into alkanes. At the same time, the photosystem AI (psbAI) promoter which is activated under white light upregulates transcription of every component in the FASyn operon which rapidly produces and elongates fatty acids in the cell. At night, the lux components will be the dominant light source in the cells, and these components produce blue light. Under the influence of blue light (and only blue light), the psbAII promoter is activated and upregulates the production of the FAP enzyme and thereby the conversion of the long-chain fatty acids into alkanes. At the same time, the psbAI promoter is inactive under the influence of blue light only which prevents further transcription of the FASyn components at night.
The Exporter operon naturally operates as a passive importer protein with an alkane-philic domain in its beta-barrel transmembrane subunit. Because alkanes are toxic to the cells, the buildup of alkanes in the cells would limit growth rates of the cells. Furthermore, the harvesting of said alkanes would require killing the cells to extract the alkanes they have produced. The purpose of the Exporter operon is to facilitate the motion of the alkanes across the membrane and out into the extracellular matrix. Although the AlkL protein functions as an importer, its passive nature makes it a prime candidate for directed evolution in the future to "flip" the function of this protein into a passive exporter. This would enable harvest without needing to kill the cyanobacteria which would allow for continuous production rather than batch production. Furthermore, exporting alkanes into a polar medium (water) would cause the low density alkanes to rise to the top of the solution which lowers the alkane content of the extracellular matrix leading to a decrease in toxicity for the cells.
Thus, the system produces long chain fatty acids during the day, and converts those fatty acids into alkanes at night. As alkanes are being produced, they are exported into the extracellular matrix and rise to the top of the solution where they can be easily siphoned off during harvest.
Another design feature is the ParB plasmid. The ParB plasmid was a critical addition to our design. Introducing a ParB plasmid would essentially remove the necessity of antibiotic selection in our system as it would work to ensure faithful segregation of the low-copy number plasmids. This would limit the effects of genetic drift in our continuous production scheme which was an issue Nathan Tague foresaw with our system. The parB plasmid contained two PCR products that needed to be Gibsoned together. One product targeted a section of the Fas1 template by using a VF2 and parB-A primer. The other product used the Synechococcus sp PCC 7002 DNA as template with parB-R and parB-F primers.
