why address arsenic?
Clean water is essential to all living things! This is especially true in a desert climate like Arizona. About 5% of the state's population gets their water from domestic wells, but they are not regulated by any government agency (1). Because these wells are private, they need to be maintained by the well owners, which can be a daunting task. Arsenic contamination in groundwater is a serious problem in local rural communities: ~20% of wells in Arizona exceed the safe concentration of 10ppbm (parts per billion) [1,2]. This contamination is a result of extensive mining in the area, and prolonged exposure to arsenic contamination can cause:
- Muscle weakness and cramping
- Negative impacts on cognitive development
- Skin rashes and lesions
- Vascular damage
- Liver failure [1,3]
Because so many Arizonans rely on well water, it is imperative that we design a long-lasting, efficient solution so that they can guarantee that the water they drink is safe and clean.
goals and progress
This project aimed to engineer the microalgae Chlamydomonas reinhardtii to clean arsenic out of water. We introduced three genes into the chloroplast of Chlamydomonas reinhardtii to biologically bind water-soluble arsenate, reduce it, and sequester the toxic metal, producing cleaner, safer groundwater. In addition to developing a system that remediates arsenic from drinking water sources, we have also modeled and prototyped a filtration for proposed implementation.
why choose chlamydomonas?
This experiment builds on previous iGEM projects and other experts’ work to engineer the chloroplast of C. reinhardtii. The microalgae is a perfect fit for our project because of its growth conditions and previously identified abilities for the sequestration of metals. It can be grown in wastewater, which makes it an ideal bioremediation material due to its ability to participate in the recycling process and avoid generating further waste (Hasan et al. 2014). Additionally, it was found to efficiently sequester uranium, another metal that has polluted many areas (Baselga-Cervera et al. 2018). Previous studies have also revealed the ability of Chlamydomonas to have increased bioremediative metal tolerance for cadmium and zinc upon genetic engineering (Ibuot et al. 2020).
The plasmid used in this project, pASapI, is designed to integrate upstream and downstream of the photosystem II sequence (psbH) in the chloroplast and was transformed into a mutant selection strain of C. reinhardtii CC-4388. Selection strain C. reinhardtii is lacking photosystem II and cannot photosynthesize. Integrating pASapI rescues function, allowing the transformed algae to photosynthesize as a selective trait . Using standard molecular cloning methods metallothionein , arsenate reductase , and ferritin  were integrated into the pASapI plasmid in different permutations.
These sequences were taken from previous iGEM teams and public databases, codon-optimized for expression in the C. reinhardtii chloroplast and synthesized. Metallothionein, a small thiol-rich metal-binding protein, binds arsenite, As(III),  while ferritin, a spherical protein forming a nanocage, binds arsenate, As(V) . Arsenate reductase catalyzes the reduction of As(V) to As(III) allows for binding with both metallothionein and ferritin to occur in the chloroplast.
We used two methods of transformation in this project: biolistic bombardment using DNA coated high-velocity tungsten particles  and agitation of an algal/DNA suspension using glass beads .
Upon successful cloning of the algae, absorption was tested at the following concentrations of arsenic: 0ppb, 50ppb, and 500ppb. These are concentrations reflective of those found in our state and provide information regarding the success of our added genes in improving the capabilities of bioremediation in Chlamydomonas.
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- Wannathong, T., et al., 2016. Applied Microbiology and Biotechnology, 100(12), pp.5467-5477.
- Chlamydomonas Resource Center, 07-Dec-2016. [Online]. Available: https://www.chlamycollection.org/. [Accessed: 03-Sep-2021].
- N. C. Keen, “Bba_k3275000 Arsenic Metallothionine,” Registry of Standard Biological Parts, 12-Sep-2019. [Online]. Available: http://parts.igem.org/Part:BBa_K3275000. [Accessed: 03-Sep-2021].
- “Arsenate reductase,” UniProt ConsortiumEuropean Bioinformatics InstituteProtein Information ResourceSIB Swiss Institute of Bioinformatics, 02-Jun-2021. [Online]. Available: https://www.uniprot.org/uniprot/P0AB96. [Accessed: 03-Sep-2021].
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- A.-M. Sevcenco, M. Paravidino, J. S. Vrouwenvelder, H. T. Wolterbeek, M. C. M. van Loosdrecht, and W. R. Hagen, “Phosphate and arsenate removal efficiency BY thermostable ferritin enzyme from Pyrococcus furiosus using radioisotopes,” Water Research, vol. 76, pp. 181–186, Jun. 2015.
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