title:'Cycle 1: Initial Trial of Full Construct (GA 1-10)',
phases:[
{
title:'Design',
description:`
<p><b>Nitrate Reduction Pathways</b></p>
<p>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).</p>
<p><b> Construct </b></p>
<p>The plasmid construct consists of 8 fragments, which include a promoter, six genes with their corresponding RBS sequences, and a terminator. The total length of the insert, excluding the plasmid backbone, is 10,168 base pairs. In order to facilitate synthesis and assembly, the fragments have been divided into four gBlocks of approximately 3,000 bp each. Each gBlock has been designed with overlapping sequences (overhangs) required for Gibson assembly to ensure precise and efficient integration of the fragments into the pSEVA261 vector backbone. Prior to assembly simulation in SnapGene, the sequences of all fragments were codon-optimised with Benchling's codon optimisation tool and verified with a personal codon frequency table obtained from (Source) to enhance translational efficiency and allow for proper gene expression in V. natriegens.</p>
<p>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.</p>
