improvement of a part
Computationally, we built and codon optimized thirteen parts which can be found on our Parts page. While only three of them were fully transformed into C. reinhardtii, over half of them were successfully cloned into recombinant E. coli cells.
Importantly, we drew on a previous iGEM team’s work to create our metallothionein, part BBa_K4006002. RHIT’s 2019 iGEM team had introduced the human gene for arsenic(III)-targeting metallothionein into E. coli cells with relative success in reducing the concentration of arsenate ions out of an aqueous solution. Given Chlamy’s natural predisposition to accumulating arsenic and over heavy metals, we decided to incorporate their gene into the chloroplast of our chassis, hoping for even further improved uptake ability.
Using the open source tool Chlamy Sequence Optimizer, we were able to codon optimize their part for use in the chloroplast of C. reinhardtii. We also created a version of this gene tagged with a polyhistidine tag. Both of these constructs were successfully transformed into the microalgae, and we were able to evaluate their abilities to accumulate arsenic in comparison to the wildtype. As seen below, both the untagged (indicated C1B) and tagged metallothionein (C3B) did show a relatively increased value in the arsenic sequestration rate, but only the tagged metallothionein was significantly increased from the wildtype (WTB). Further information can be found on our Results page.
creation of new parts
The other parts created were based mainly off of two basic parts: arsenate reductase and ferritin. Arsenate reductase catalyzes the reaction of As(V) to As(III). While MT mainly targets As(III), ferritin is more prone to sequester As(V). Arsenate reductase allows both proteins to bind to the arsenic effectively. This part was newly created by us, and sourced from a known arsenate reductase sequence and codon optimized. The tagged version of this construct was successfully transformed into C. reinhardtii, and the arsenic uptake experiments indicated a significant increase in the ability of this strain (C2B) to sequester arsenic in comparison to the wildtype (WTB).
We have also spent time identifying methods to express numerous genes in a single plasmid, as there are little tools available to do this in C. reinhardtii. Intercistronic expression elements have been identified that seem to be able to link numerous genes in a plasmid and allow them to express concurrently. However, they are not well classified, and experts have suggested that not all of them are effective. We detail this exploration further in our Engineering Success page.
methods of transformation
While transformation into E. coli and other model organisms are well characterized, there are few reliable methods for microalgae like C. reinhardtii. In this project, we learned and used two of these methods: biolistic transformation and glass bead transformation. As detailed on our Collaborations page, we used input from other teams in our protocols for the biolistic transformation and detailed our experiences with both methods in a collaborative phototroph handbook.