Towards engineering Chlamydomonas reinhardtii for botryococcene synthesis
Concerns about climate change and rising prices of petroleum-based fuel have sparked a search for sources of renewable energy. The microalga Botryococcus braunii is a potential source as this species produces botryococcenes, hydrocarbons that can be converted to combustible fuels. However, due to its slow growth rate and lack of molecular toolkit, this algal species is not a feasible source of commercial biofuel. The Stern Lab at BTI is performing research to address difficulties associated with microalgal biofuel production. As an intern, my goal was to engineer the fast-growing microalgae Chlamydomonas reinhardtii with the ability to produce botryococcenes. Using primarily PCR and a cloning technique called Gibson Assembly, I generated constructs containing 1) regions of the C. reinhardtii chloroplast genome to promote integration of foreign genes via homologous recombination and 2) genes for botryococcene synthesis including or excluding the botryococcene methyltransferase gene. Chloroplasts of a mutant C. reinhardtii strain containing a truncated rbcL gene, whose function is required for photosynthesis, were transformed with these constructs using particle bombardment. Transformants were isolated based on their ability to perform photosynthesis due to incorporation of the complete rbcL gene in the constructs, and were subsequently analyzed for transgene insertion using PCR and DNA gel blots. The transgenic algae will be analyzed for botryococcene production and if accumulation is confirmed, the system will be adopted for Chlorella, an algal species more suitable than Chlamydomonas for commercial purposes. Therefore, this research may help establish a method to produce commercial microalgal biofuel at a reasonable cost.
My BTI internship this summer confirmed my desire to attend graduate school and pursue a career in scientific research. I learned many new laboratory techniques, such as chloroplast transformations, Gibson Assembly, and various DNA preparation protocols. I also became comfortable with techniques to which I had previously been introduced but could not have performed completely independently or efficiently, such as PCR, agarose gel electrophoresis, and restriction digests. Working in a laboratory for ten weeks has also greatly enhanced my critical thinking skills and ability to construct a research plan, take the steps needed to reach my goals, and make changes to my plan according to results. This internship has given me an idea of what graduate school or a career as a scientific researcher could mean for me. In addition to this, BTI has a very friendly and comfortable atmosphere, and was truly an enjoyable place to be every day!