Though not a continuation of our 2021 UMaryland project, our project builds off the mission of that project: to decrease nutrient pollution in the Chesapeake Bay.
After doing a review of existing iGEM nitrate remediation projects, we realized most of the projects focus on soil or prioritize the conversion of nitrate to nitrite to ammonia (2017 iGEM NCKU_Tainan and 2020 iGEM Lambert). Our system will contribute to other iGEM teams by being oriented around removing nitrate from bodies of water.
Our genetic construct will be useful to future teams because the modularity of genes is embedded in the design. Our construct uses Gibson Assembly and Golden Gate, creating a roadmap for future teams to easily swap and test genes. Gibson Assembly is used to ligate large genes that were synthesized as separate gene blocks together. This allows the testing of individual genes before testing how the genes work in tandem with one another. Each gene can be cloned into a single plasmid via Golden Gate. This modular design can be used by future iGEM teams, specifically with large bp constructs synthesized by companies like Integrated DNA Technologies.
We also contribute to future iGEM teams by conducting a literature review that investigates how to create a nitrogen sink to drive forward endogenous pathways in E. coli to uptake nitrates. Here are some of the findings from our literature review:
Researchers have long sought to remove or sequester nitrogen such as nitrates and ammonia from our environment. However, the driving motivation was not always in chasing a lower level of nitrogen. Instead, many nitrate uptake and assimilation studies were motivated by the need for more resilient and higher-yield crops, which ultimately requires them to uptake more nitrogen. There have been ample studies that genetically modify varying aspects of the glutamate dehydrogenase and the glutamate synthase/GOGAT pathways—to just list a few— in both bacteria and plants, Both pathways are intimately involved in assimilating nitrogen into the organism’s biomass by converting and storing them in amino acids and polymeric proteins. There has also been significant engineering on the nitrate reductases that are involved in reducing nitrate into a much more usable form of nitrogen in ammonia, also in an effort to increase crop yield (Lebedev). Though this paper’s focus is on plants, it is important to note that almost all living organisms have analogous systems to those mentioned earlier. Regardless, the spirit of this paper is very in line with our goal of improving nitrate uptake and storage as well even though the end goal may differ slightly.
Regarding CGP synthetase, there are studies from more than two decades ago asking very similar questions to what we are trying to answer through our engineering today; that question is how we can co-opt and engineer CGP synthetase to competitively meet some market needs or address a societal challenge. By standing on the shoulders of their work and progress, we now strive to overcome our own challenge of nitrogen pollution in the bay. In an early CGP synthetase paper published by Aboulmagd et al. in 2000, they not only characterized the cphA gene encoding CGP synthetase in its native cyanobacteria Synechocystis, but they also engineered a recombinant E. coli with the same cphA gene to characterize the viability of heterologous cphA expression compared to that of homologous expression under varying conditions (Aboulmagd). Another paper published more recently in 2021 by Kwiatos et al. delves deeper and expands upon the many potential applications of CGP synthetase that were not feasible back then, including scaling up production to use CGP as a starting material in manufacturing, transforming a wider variety of organisms with the cphA gene, and modifying the enzyme itself for increased efficacy (Kwiatos).