<Nav.Linkas={Link}to="section1"smooth={true}duration={500}className={activeLink==='section1'?'active':'notActive'}>ODE Model of Biochemical Reactions</Nav.Link>
<Nav.Linkas={Link}to="section2"smooth={true}duration={500}className={activeLink==='section2'?'active':'notActive'}>Metabolic Engineering Strategy to Reduce Ammonia Production</Nav.Link>
<p>The Michaelis-Menten mechanism describes the enzymatic conversion of a substrate <MathJax.Provider>
<span>
...
...
@@ -187,7 +188,8 @@ export function Model() {
<MathJax.Provider>
<divclassName='indent formula_content'>
<spanclassName='formula_line'><MathJax.Nodeformula={`{{E+S}\\overset{k_{f1}}{\\underset{k_{r1}}{\\rightleftharpoons}} ES \\xrightarrow[]{k_{cat}}E + P}`}/></span>
<p>The FeaR-phenylacetaldehyde complex can bind and activate the PTynA promoter. In this case, the concentration of the enzyme-substrate complex <MathJax.Provider>
<p>For an enzyme-catalyzed reaction involving two substrates that form a single product, the Michaelis-Menten equation can be extended to account for the involvement of both substrates. The general reaction can be written as</p>
<p>TPH1 converts tryptophan that enters the cytoplasm into 5-hydroxytryptophan, which is then transported from the cytoplasm into the gut. The corresponding reaction equations and ODEs are as follows</p>
<p>To identify knockout targets, we ran FBA optimization on iDK1463, yielding the flux values for various metabolic reactions in the optimized strain. Next, we filtered out the reactions related to ammonia, excluding those with a flux of zero under normal physiological conditions. The biomass reaction and reactions linearly related to it were considered essential and not selected as targets. Ultimately, among the <b>6 reactions identified</b>, the flux of ammonia-producing reactions will be minimized, while the flux of ammonia-consuming reactions will be maximized.</p>
<figcaptionclassName='caption'>Figure 2: Representation of the metabolic genotype</figcaption>
<figcaptionclassName='caption'>Figure S2: Representation of the metabolic genotype</figcaption>
</div>
</div>
</div>
<p>Based on the OptiGene algorithm, we identified two valuable target reactions: <b>Glycine Cleavage System (GLYCL)</b> and <b>Glutamate Dehydrogenase (NADP, GLUDy)</b>. The main reactions involved, the candidate knockout targets, and the effects of their knockout on flux are summarized in the table below:</p>
<p>Based on the OptGene algorithm, we identified two valuable target reactions: <b>Glycine Cleavage System (GLYCL)</b> and <b>Glutamate Dehydrogenase (NADP, GLUDy)</b>. The main reactions involved, the candidate knockout targets, and the effects of their knockout on flux are summarized in the table below:</p>
<pstyle={{color:'#656563'}}>Table 2: Candidate knockout target genes identified by OptGene</p>
<tableclassName="model-table three-line-table">
<caption></caption>
<thead>
<trclassName='table-head-line'>
<th>Target Reaction</th>
...
...
@@ -962,12 +1058,13 @@ export function Model() {
<p>Subsequently, we evaluated the potential impact of <b>15 candidate genes</b> resulting in <b>32,767 knockout combinations</b> on the growth and ammonia production of the strain using FBA. The results indicated that enhancing the reverse reaction of GLUDy is a key factor in reducing ammonia production, while the knockout of the <b>ECOLIN_RS15500</b> gene (corresponding to the ENO reaction) is a crucial step toward achieving this goal. Although the knockout of the GLYCL reaction can also reduce ammonia production to a small extent, it is not a primary factor. Additionally, different knockout schemes significantly affect the strain's growth, specifically the biomass flux.</p>
<p>Considering that excessive knockouts may impair the strain's normal physiological functions and increase operational complexity, we ultimately identified <b>6 alternative knockout schemes</b> that balance ammonia production and biomass:</p>
<pstyle={{color:'#656563'}}>Table 3: Knockout schemes we ultimately selected.</p>
<tableclassName="model-table three-line-table">
<thead>
<trclassName='table-head-line'>
<th>Target Genes for Knockout</th>
<th>Biomass Flux [mmol/(gDW*hr)]</th>
<th>Ammonia Production per Biomass [mmol/(gDW*hr)]</th>
<th>Ammonia Production Unit Biomass [mmol/(gDW*hr)]</th>
<th>Biomass Ratio Compared to WT (%)</th>
<th>Ammonia Production Ratio Compared to WT (%)</th>
</tr>
...
...
@@ -985,6 +1082,11 @@ export function Model() {
</tbody>
</table>
<p>The table displays the biomass flux and ammonia production per unit biomass for the wild-type strain and various knockout strains, along with the percentage of these values compared to the wild-type strain.</p>
<figcaptionclassName='caption'style={{paddingRight:'20%'}}>Figure 1: Growth of Escherichia coli Nissle 1917 in M9 and LB Medium</figcaption>
<p>We compared the changes in biomass flux before and after gene knockouts predicted by the model with the growth characteristics of *Escherichia coli* Nissle 1917 in M9 and LB media obtained from wet experiments. After gene knockout, the biomass flux of the strain reached <b>80.03%</b>of its pre-knockout level under optimal conditions. In the M9 medium, which inhibits ammonia production, the growth rate of the strain was approximately <b> 81.77%</b> of that in LB medium. This validates the model's calculations and demonstrates that merely inhibiting ammonia production does not significantly affect the strain's growth, indicating the feasibility of the gene knockout strategy.</p>
<p>In summary, our metabolic engineering analysis provided multiple knockout schemes, including single and multi-gene knockouts. Through gene knockout, ammonia production can be reduced to approximately <b>18% </b>of the wild-type levels, while the growth rate of the strain can be maintained at about <b>80%</b> of the wild-type. This finding offers significant directions for genetic modifications in the development of engineered strains intended for in vivo therapy.</p>
<pclassName="indent">We have built several BioBricks, which cover all aspects throughout our projects, constituting our sensory module, metabolic module, and safety module.</p>
<h2>1.Our Basic Parts</h2>
<p>We designed six basic parts in total this year,you can click on the part to see details.</p>
