Water is a basic need that people in our state of Arizona still struggle to access due to the state’s long history of water scarcity and contamination. Heavy metal contaminants, notably arsenic, pervade the groundwater supply in Arizona due to decades-long unregulated mining activity. A study conducted by the US. Geological Survey in the main aquifers in the southwestern United States found that about 19% of the drinking water wells sampled exceeded the maximum contaminant level for arsenic of ten micrograms per liter established by the US Environmental Protection Agency ^{[1, 2]}. Arsenic contamination in water threatens the health and well-being of rural Arizonans who rely on these water sources.

ASU iGEM decided to tackle this problem by creating Chlamydomon[As], an engineered microalgae capable of bioremediating arsenic from contaminated water. Chlamydomonas reinhardtii has the ability to naturally uptake arsenic on its own by binding arsenite (As(III)) that enters the cell to metal binding peptides called phytochelatins, which bind to the arsenite and sequester it in the vacuole of C. reinhardtii . We are taking advantage of this existing biological pathway by introducing the genes encoding for the enzymes arsenate reductase (ACR2p) and phytochelatin synthase (PCS) into C. reinhardtii . Arsenate reductase reduces incoming arsenic into the cell into arsenite, the kind that can bind to phytochelatins. Phytochelatin synthase is the rate-limiting enzyme involved in the production of phytochelatins, and by increasing its expression, we can increase phytochelatin synthesis in C. reinhardtii , thereby enhancing its ability to bind and sequester arsenite in the vacuole. Chlamydomon[As] has the potential to revolutionize bioremediation technologies to help Arizona and regions around the world solve the issue of arsenic contamination in water.

A project like Chlamydomon[As] requires many moving parts to come together to forge a unique and innovative solution, but also presents formidable challenges.

At the cellular level, nuclear expression of transgenes in C. reinhardtii poses a number of obstacles that must be taken into account in order to achieve success. Firstly, transgenes are inserted randomly into the nuclear genome, meaning that strong expression of genes of interest is not guaranteed. Second, exogenous DNA is often not integrated fully or as intended into the nucleus, meaning that further regulation is required in order to maintain the construct’s integrity ^[3]. In an attempt to mitigate some of these challenges, we chose to develop a tricistronic expression vector that would allow us to express our two genes of interest (ACR2p, PCS) and an antibiotic selection marker gene in the C. reinhardtii nucleus, all under the control of a single promoter using viral 2A peptide sequences ^[4].

More information about our novel multicistronic expression vector is below.

Implementation also presents itself as a challenge. Although C. reinhardtii has long been well-characterized and considered a model organism for laboratory work, this means that the specific conditions that allow it to thrive in the laboratory must be replicated in order for it to survive outside of the lab as well. Our water filter design was designed to account for temperature regulation, growth times, and heavy metal uptake rates of the algae.

Learn exactly how we overcame these design constraints on our implementation page.

In order to overcome the challenges presented by transgene expression in the C. reinhardtii nucleus, we chose to develop a tricistronic expression vector that would allow us to express our two genes of interest (ACR2p, PCS) and an antibiotic selection marker gene (paromomycin) in the C. reinhardtii nucleus, all under the control of a single promoter.

This system is significant because engineering the C. reinhardtii nucleus was previously considered to be limited to 2 genes of interest [4]. Our system, however, introduces the opportunity to add an additional gene of interest to the plasmid, which greatly increases the possibilities of engineering in C. reinhardtii . We conducted preliminary testing a total of 6 2A peptides to determine which had the highest cleaving efficiency. Future efforts can use the most efficient 2A peptide to construct a more efficient version of Chlamydomon[As].

This data can also be used to construct a template plasmid that can accommodate up to three genes of interest using this 2A peptide system. Parts like these will enable future iGEM teams working with C. reinhardtii the ability to more accessibly develop ambitious genetic constructs.

View our contributions to the iGEM Parts registry here.

Chlamydomon[As] represents a small portion of the efforts being made to combat water quality issues with bioremediation. We are proud to not only have contributed to research in methods of bioremediation, but also have advanced research in engineering C. reinhardtii itself. Though there is still a long way to go until bioremediation technologies are normalized for public or commercial use, we hope that our project sheds light on how important these solutions are and broadens the horizons of what was previously thought to be possible in the field of water bioremediation.