In this section, we walk through our design cycles.
# Inspiration & Initial Project(s)
The search for a project topic and goal began once the team was assembled in February, with many ideas put under consideration and discarded over the course of dozens of meetings, literature review and brainstorming.
In keeping with the tradition of the MSP iGEM teams of the past years, the team chose to focus on combating a local problem.
Following weeks of brainstorming, the team turned to work on the development of a concept for the synthesis of a biocellulose-chitosan co-polymer. We envisioned this co-polymer could be used to make clothes for astronauts, with functional proteins and enzymes bound to the surface to provide antimicrobial properties. This would ensure a healthier living environment in habitation in space, as well as cut down on the waste and odour of disposable clothes.
This idea evolved into functionalising the co-polymer as a filter and film, and later granules, used for water treatment and filtration, which could double as a fertiliser through recycling into compost. This shift arose as a reaction to several issues of feasibility and efficiency we found with the initial idea, as well as a push to make the idea more local.
Eventually, the co-polymer concept was discarded, and the team focused on trimming down and finalising details for a concept that could be a winner.
Finally, the team committed to a concept outline and began work on literature review and background research to prepare for entering the lab work phase of our project. *Vibrio natriegens* was selected as a chassis organism, with the combating eutrophication being selected from the several water treatment focuses possible.
# Purpose
The purpose of the wet lab design was driven by the team’s inspiration, tackling coastal eutrophication with a circular and sustainable approach. Natronaut’s aim is to restore ecosystem balance, mitigating the hypoxic effects of nitrate (NO3-) induced algal bloom and decomposition (Cosme and Hauschild, 2017; Ærtebjerg, 2001). Considering a fully circular approach, the team decided to further extend the project’s purpose to the prevention of eutrophication development. This would be done by recycling the cells into single-cell proteins, which would supplement animal feeds and lower fertiliser use in traditional animal feed production.
# Engineering Design & Planning
## Selection of Chassis Organism
After finalising the main goals for the project, the team focused on finding the perfect chassis. *Vibrio natriegens* (*V. natriegens*) was deemed the most suitable choice and selected as the chassis organism for the Natronaut project.
Multiple factors played into this decision, but the main inspiration came from a previous iGEM team: the Marburg 2018 *VibriGens.* This team engineered the wildtype strain *ATCC14048* to make it more suitable for research by optimising the strain for cloning, protein expression, and protein interaction studies (Marburg, 2018).
In addition to the availability of an optimised toolkit for this organism, it was selected in alignment with the purpose of the project: combating coastal eutrophication. A resilient and fast-growing organism that could withstand the high salinity of this environment and provide the increased uptake of nitrates was optimal (Thoma & Blombach, 2021).
Additionally, its ability to grow on cheap carbon source media, ability to pump proteins into its medium, and finally, the surge in interest for it in the synthetic biology field in the last few years made it an even more appealing option (Weinstock et al., 2016).
With its robust growth and protein expression capabilities, Natronaut aims to contribute to establishing this strain of *V. natriegens* as a key chassis for synthetic biology, expanding on the *VibriGens* team’s research (Marburg, 2018).
## Designing of The Biological System
**Nitrate Reduction Pathways**
Nitrates can be removed from water through several bacterial metabolic processes. The most prevalent pathway is denitrification, in which NO3- is sequentially reduced to NO2- and then to N2, which is released into the atmosphere (Zhao et al., 2018). Other important nitrate reduction pathways include dissimilatory NO3- reduction to NH4+ (DNRA) and NO3- assimilation (Moreno-Vivián et al., 1999).
DNRA, typically utilised by bacteria in anaerobic conditions for the purposes of energy conservation, involves converting NO3- into NH4+ in a two-step reaction via the NO2- intermediate (Herrmann & Taubert, 2022). While DNRA retains nitrogen in its bioavailable form (NH4+), it does not directly incorporate it into organic compounds. Thus, both denitrification and DNRA result in the loss of available nitrogen - either as atmospheric nitrogen in the case of denitrification or as NH4+ that is not assimilated into biomass in the case of DNRA.
In contrast, the assimilatory NO3- pathway leads to incorporation of nitrogen into organic compounds, such as amino acids, conserving it within the organism (Moreno-Vivián & Flores, 2007; Jiang & Jiao, 2015). These amino acids can be then used to produce SCPs. The assimilatory pathway does not only retain nitrogen, but also contributes to the production of microbial biomass, hence providing a more efficient way of nitrogen utilisation.
The assimilatory pathway in bacteria comprises several steps. First, NO3- is captured and internalised from the extracellular environment to the intracellular space. This step is mediated by a NO3–-transporter, which is most commonly an ATP-binding cassette (ABC)-type transporter located in the cytoplasmic membrane (Moreno-Vivián & Flores, 2007). The transporter consists of three subunits: a periplasmic protein that binds NO3- with high affinity (even at low extracellular concentration of NO3-), a transmembrane protein that facilitates the transport of NO3- across the membrane, and a cytoplasmic ATPase anchored to the membrane, which hydrolyses ATP to provide energy for the process (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007).
Once the NO3- is internalised, it is then reduced to NO2- by assimilatory nitrate reductase (Nas). This enzyme is NADH-dependent, and has two subunits: a large catalytic subunit, which contains the essential active site for the reduction of NO3- and a small NADH oxidoreductase subunit, which facilitates the transfer of electrons to the active site (Lin & Stewart, 1997; Moreno-Vivián & Flores, 2007). In the following step, NO2- is further reduced to NH4+ by the monomeric nitrite reductase Nir. Afterwards, the produced NH4+ is incorporated into amino acids, specifically glutamine and glutamate through the GS-GOGAT and GDH pathways (Moreno-Vivián & Flores, 2007; van Heeswijk et al., 2013). The GS-GOGAT pathway consists of two key steps. First, the enzyme glutamine synthetase (GS) catalyses an ATP-dependent reaction that converts glutamate to glutamine by incorporating an ammonium ion. Following this, glutamate synthase (GOGAT) transfers the amide group from glutamine to 2-oxoglutarate, resulting in the production of two glutamate molecules. In contrast, the GDH pathway employs a more direct approach. The enzyme glutamate dehydrogenase (GDH) catalyses the incorporation of an ammonium ion (NH₄⁺) directly into 2-oxoglutarate, forming glutamate in a single step. The resulting amino acids, glutamate and glutamine, undergo further transamidation and transamination, yielding various amino acids, which then serve as building blocks for the biosynthesis of proteins during translation (van Heeswijk et al., 2013).
