Results

Our team learned how to grow and transform Chlamydomonas reinhardtii for the first time at our institution.

We learned how to codon-optimize gene sequences for expression in C. reinhardtii and domesticate the sequences by removing SapI, BsaI, and BbsI recognition sequences.

We also successfully designed and built the following plasmids using Golden Gate Assembly Technology - (Contribution Page)

For enhanced phosphate uptake:

L1-crnpt
L1-Psr
L2-phosphate

For denitfrification of wastewater:

L0-HAO
L0-AMO
L0-nosZ
L1-HAO
L1-AMO
L1-nosZ
L1-aadA
L2-nitrogen

We successfully transformed C. reinhardtii with the L2-phosphate plasmid and successfully confirmed the expression of the Psr-clover fusion protein.

Fluorescent microalgae under a microscope, showing successful uptake of our phosphorus pathway genes — This image shows samples of *Chlamydomonas reinhardtii* flourescing, which helps us identify which green algae successfully took up our phosphorus pathway genes. The fluorescence is caused by the Psr-clover fusion protein, which was added to our phosphorus construct to confirm expression of our protein. We were also able to confirm our L2 Nitrogen plasmid transformation and expression using fluorescence, as shown below.

This image shows samples of *Chlamydomonas reinhardtii* flourescing, which helps us identify which green algae successfully took up our phosphorus pathway genes. The fluorescence is caused by the Psr-clover fusion protein, which was added to our phosphorus construct to confirm expression of our protein. We were also able to confirm our L2 Nitrogen plasmid transformation and expression using fluorescence, as shown below.

We learned from many other individuals and entities involved with the health of Utah Lake, and incorporated their input into our overall project design.

Next steps

Determine if our engineered strains of C. reinhardtii are more efficient at removing phosphorous and nitrogen from wastewater than wild-type strains of the algae.

Identify an appropriate auxotrophic strain of C. reinhardtii to engineer with our constructs to prevent its escape in the wild.

If our next steps are successful, we could utilize our engineered algae for bioremediation. It could be used for cleaning up polluted water or soil by absorbing contaminants (Nitrogen and Phosphate). If our engineered algae is also safe for agricultural use after absorbing contaminants it could be used as fertilizer, away from water bodies.

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