@@ -21,6 +21,7 @@ Finally, the team committed to a concept outline and began work on literature re
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.
**Figure 1.** Project purpose and goals outline. Created with Biorender.com
# Engineering Design & Planning
## Selection of Chassis Organism
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@@ -47,7 +48,10 @@ The assimilatory pathway in bacteria comprises several steps. First, NO3- is cap
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).
**Figure 2.** The pathways that result in the biosynthesis of glutamine and glutamate. The GDH pathway is shown in the left panel. The GS-GOGAT pathway is shown in the right panel. Created with BioRender.com
**Figure 3.** Genes in K. oxytoca
In K. oxytoca, the genes associated with the nitrate (NO₃⁻) assimilation pathway are arranged in a nasFEDCBA operon. Within this operon, the nasFED gene cluster encodes the transporter necessary for the uptake of extracellular NO₃⁻. Both the membrane-spanning subunit NasE and the ATP-binding subunit NasD play essential roles in the acquisition of nitrate. The genes nasA and nasC encode the large and small subunits of assimilatory nitrate reductase (Nas), respectively, while the nasB gene is linked to nitrite reductase (Nir). This genetic arrangement allows K. oxytoca to efficiently assimilate nitrate (Wu & Stewart, 1998).
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For the construction of this biological system, we selected three key components of the assimilatory nitrate reduction pathway: the nitrate transporter, nitrate reductase (Nas), and nitrite reductase (Nir). These proteins are encoded by a cluster of six genes derived from Klebsiella oxytoca (K. oxytoca) (strain: M5aI) (Wu and Stewart, 1998).
**Figure 4.** nasFEDCBA operon as seen in K. oxytoca. Sourced from Wu and Stewart, (1998).
These genes were selected for two reasons. First, *K. oxytoca* and *V. natriegens* share taxonomic similarities as they are both Gram-negative bacteria of the class Gammaproteobacteria (Reimer et. al, 2022). This increases the probability of successful gene expression in the chassis organism due to the compatibility of their transcription and translation machinery and similarities in gene expression. Secondly, these genes function as a single operon in *K. oxytoca*, which facilitates their coordinated expression, as all the regulatory elements are retained and proteins produced simultaneously (Wu et al., 1998). This organisation ensures that the components of the ANRA pathway are properly expressed, potentially leading to higher efficiency than combining individual genes from different organisms.
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@@ -73,10 +78,12 @@ In previous studies on the nasFEDCBA operon in *K. oxytoca*, RBS sites were foun
Additionally, we sourced a native P1 promoter and a B0015 terminator from the Collection as well, which came together in the following sequence (Figure X.). The promoter and terminator were initially taken from Tschirhart et. al (2019) as they have previosly tested them, and have demonstrated high protein expression levels in *Vibrio natriegens.*
**Figure 5.** The sequence of genes, including the promoter, terminator, and RBSs selected from the Marburg Collection
To ensure the correct folding of the proteins encoded by the identified genes, their three-dimensional structures were simulated using AlphaFold2, an artificial intelligence tool with high accuracy of predicting protein structures based on primary sequences (Yang et al., 2023). Simulations were conducted using the AlphaFold2 Colab notebook. The amino acid sequences (represented in one-letter code) for each protein were entered into the AlphaFold2 pipeline as input. The predicted protein structures were then downloaded in PDB format and analysed using the molecular visualisation software ChimeraX-1.8. The resulting structures of the enzymes and the transporter are illustrated below.
**Figure 6.** Proteins of interest. Simulated in AlphaFold2, visualised using ChimeraX-1.8.
The complete sequence of the genes and genetic parts was then divided into fragments smaller than 3000 base, for ordering, with overhangs added for Gibson Assembly.
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After thorough research on the subject, the team decided to opt for plasmid pSEVA261 with a p15a origin of replication due to its high maintenance, high stability, and low copy number in *Vibrio natriegens* (Tschirhart et al. 2019).
**Figure 7.** pSEVA261 plasmid. Image taken from SnapGene.
The team chose to have a low copy number plasmid for this design, as Tschirhart et al. proved that a low copy number did not lead to low maintenance in this case, and overburdening the cells with over-production of such a large plasmid was deemed undesirable.
