Unicellular microalgae are a varied group of organisms with excellent potential in applied and exploratory synthetic biology. Compared with the majority of other model and industrial organisms, most microalgae have the environmentally beneficial distinction of being able to carry out photosynthesis. With their photosynthetic and mixotrophic abilities, these organisms have the promise of becoming platforms for carbon-neutral production of both high value and inexpensive metabolites. Additionally, microalgae have the capability to express genes from plants, fungi and prokaryotes making them ideal hosts for combinatorial recombinant gene expression.

Non-engineered strains are already being used for biofuels and lipids. With the creation of standardised tools for the stable engineering of microalgae, the community will be able to continue asking deeper questions about basic biology and, drastically increase the promise of microalgae as an industrial chassis.

By building and characterising tools for genome scale engineering of multiple genera of microalgae, the Concordia iGEM team has opened a new world for the use of industrially relevant photosynthetic microbes by iGEM teams and researchers alike.

Starting with the goal of making microalgae a viable platform in synthetic biology, we set out to create a toolkit of standardised biological parts for use in these organisms. We chose to work with a diverse group of species that included Chlorella vulgaris, Chlorella ellipsodia, Chlorella saccharophilea, Chlorella kessleri, and Chlamydomonas reinhardtii. These species present a broad range of optimum growth conditions and metabolic profiles.

Characterised parts in our toolkit include promoters, terminators, fluorescent proteins, localisation tags, antibiotic markers, and CRISPR/Cas.

Our promoter library offers plant, fungal, and viral promoters of varying strengths for control of heterologous gene expression in microalgae. Our terminators were compared alongside a variety of these promoters. Multiple localization signals were investigated to enable a user to perform targeted gene expression. We also systematically characterised the effective concentration ranges for antibiotic use in a manner not yet seen for microalgae.

CRISPR/Cas systems have revolutionized genome engineering efforts by providing a quick and efficient means of inserting and deleting DNA from the host’s genome. Our toolkit set out to demonstrate the ability to perform targeted multi-plex genome engineering of microalgae in a manner accessible to any researcher.