Engineering Success

Our Design, Build, Test Cycle

During the course of the project our team worked to follow the design, build, test, learn, cycle. Several examples of the cycle during our project are listed below.

Example 1

Design

We initially planned out a pathway for reducing nitrogen in wastewater by absorbing organic nitrogens and converting them to nitrous oxide

Build

As we began to put this plan into motion, we started to organize our plasmids.

Test

We communicated our idea to an academic working on this same problem from BYU, and he informed us that Nitrous oxide is actually a very dangerous greenhouse gas.

Learn:

We researched more and found an additional gene that would allow us to reduce Nitrous oxide to Dinitrogen gas, a non-toxic completely safe waste product. We added this gene to our pathway to prevent Nitrous oxide from being released.

Example 2

Design

Our team identified a local problem, that nutrient loading in Utah lake is causing toxic algal blooms. We researched and identified a method for our team to help address this problem by engineering a microalgae to increase its natural nutrient uptake. Our notebook and contributions page outline the specific genes and how we obtained them.

Build

Once we identified and ordered these genes, we used golden gate cloning protocols to build the all, as shown in our notebook.

Test:

When it was time to run diagnostic digests of our level 2 Nitrogen plasmid, we found that it was not working. There were missing bands of DNA in our gel results. We were confused, because we had thought our level 1 ligations had been successful.

Learn

We sequenced our level 1 parts and found that the L1-HAO assembly had actually been unsuccessful, so we went back and re-designed our L1 ligations, which, when implemented, led to a successful diagnostic digest of the HAO gene. Our L2 ligation was also successful, as shown below.

Example 3

Design

To transform our Chlamydomonas reinhardtii we decided to use a Neon electroporator to insert our DNA.

Build

We obtained a kanamycin resistance L1 plasmid from the Addgene Plant MoClo kit and started testing to see if we could transfect our C. reinhardtii with this plasmid.

Test

We did many trials to insert our kanamycin resistance plasmid into C. reinhardtii, but these attempts were unsuccessful. All of our microalgae would die when exposed to the antibiotic kanamycin.

Learn

We realized that a potential cause of the unsuccessful transformation could be the codon bias that C. reinhardtii exhibits, favoring G/C pairings. We were aware of this ahead of time, so we had designed our genes to be codon optimized. Due to this, when we had a functional phosphorus L2, which also had C. reinhardtii specific regulatory elements, we decided to try it in the transformation instead of the plant MoClo plasmid, and it actually worked! It expressed the kanamycin resistance, and even more than that, we had a fluorescent tag on our protein. We were able to see that fluorescence in C. reinhardtii on a fluorescent microscope.

Our left image above shows successful transformation for our phosphorus construct through fluorescence, while the right image shows the same for our nitrogen construct.