diff --git a/docs/.vuepress/components/IterativeCycle.vue b/docs/.vuepress/components/IterativeCycle.vue
index 5045920f855999d2026795d01de84e4c3e8d7d12..be3f23e50741d440f615e42581c1ac5ee4d98755 100644
--- a/docs/.vuepress/components/IterativeCycle.vue
+++ b/docs/.vuepress/components/IterativeCycle.vue
@@ -156,8 +156,9 @@ export default {
{
title: 'Module 2: Characterization',
description: `
- <p>This module covers the characterization of the assembled constructs.</p>
- <p>It focuses on experimental design, setup, and data collection.</p>
+ <p>While developing the chemical pathway and genetic design of engineered organism, a method was considered in order to verify the working of the GMO. </p>
+ <p>The functionality and efficiency of the engineered V. natriegens can be tested by probing the efficacy of the implanted enzymes. Colour reactions coupled with spectrophotometry is an effective way to establish the concentration of the various analytes. </p>
+ <p>However, due to the time constraints and difficulties with the assembly of our construct (See above), the verification and characterisation of the functioning of the pathway inserted into V. natriegens was not able to be carried out. </p>
`,
cycles: [
{
@@ -166,8 +167,32 @@ export default {
{
title: 'Design',
description: `
- <p>Designing experiments to characterize the assembled constructs.</p>
- <p>This includes defining parameters and expected outcomes.</p>
+ <p>Chemical compounds are able to absorb and refract light at different wavelengths depending on their structures, more precisely because of their ability to delocalise their π-electrons. The more freedom a π-electron has to move around the structure, the shorter the wavelength it can absorb, and the longer the wavelength which can be refracted. The ability of a compound to delocalize its π-electrons is determined by the consecutive overlap of multiple p-type orbitals generated by the presence of consecutive double (or triple) bonds. Compounds with an high degree of conjugation, like Beta-carotene (Fig. 1 Beta-carotene) absorb light in the blue region of the visible light spectrum (~450 nm) and refract on the opposite side of the spectrum, the red region (~750 nm), giving molecules their characteristic colours. </p>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/beta-carotene-2d-skeletal-svg.webp" alt="Beta Carotene" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 17. Beta-carotene. Image sourced from Wikipedia</figcaption>
+ </figure>
+ <p>This concept can be used to generate highly conjugated molecules using the target compound that has to be quantified as the starting material using a spectrophotometer and the Beer-Lambert law. This mathematical model allows the calculation of the concentration of a compound depending on its light absorbance.</p>
+ <p>Two tests have been selected for our quantification protocols, the Griess test and the indophenol blue test. </p>
+ <p>The Griess test is used for the detection and quantification of nitrates, and will be able to tell us if the nitrate reductase enzyme works. The Griess test is based on the diazotization reaction of sulfanilinic acid, where the nitrite will react with the amino moiety on the phenyl ring to form a diazonium salt intermediate. The diazonium salt is extremely electrophilic and will readily react with the ring of an aromatic compound. Depending from the aromatic compound chosen, different colours can be obtained, the most common are pink (given by 1-naphtylamine), orange (given by 1-naphtol) or yellow (given by phenol). This azo coupling reaction makes use of the nitrite substrate as a nucleophilic reagent to yield a highly conjugated system that extends from the sulfanilinic end to the napthaline moiety, able to absorb very short wavelengths and resulting in bright and warm colours (Griess reaction).</p>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/griess-test-reaction-svg.webp" alt="Griess Test" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 18. Griess test reaction scheme. Image sourced from Wikipedia</figcaption>
+ </figure>
+ <p>The indophenol blue test is used to detect the presence of ammonia to check the functionality of the nitrite reductase. It exploits the electrophilicity of the ammonium ion to link to phenol molecules, thus creating a conjugated system that is able to absorb in the red region of the visible light, yielding a deep-blue dye. The reaction starts by treating a sample suspected to contain ammonium with methanol, keeping the ammonia in its cationic form, and sodium hypochlorite to convert the ammonium in chloramine gas, which will stay dissolved in the methanol solution. The chloramine in methanol solution is then added to a mixture containing phenol to yield a chloramine to phenol molar ratio of 1:2 and sodium nitroprusside, which will serve as a catalyst. The reaction mixture then turns blue to signal that it has come to an end (Indophenol reaction).</p>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/indophenol-reaction-scheme.webp" alt="Indophenol Test" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 19. Indophenol blue reaction scheme. Image sourced from Sasongko, A. (2018).</figcaption>
+ </figure>
+ <p>The concentration of both nitrites or ammonia can then be measured by measuring the absorbance of the solution and comparing to a previously made calibration curve to know exactly the amount of substrate measured. </p>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/beer-lambert-s-law.webp" alt="Beer Lambert Law" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 20. Beer-Lambert law. Image sourced from Bhuyan, S., (2024)..</figcaption>
+ </figure>
`
},
{
@@ -184,7 +209,7 @@ export default {
{
title: 'Module 3: Integrative Vector',
description: `
- <p>In the process of developing our assembly and transformation plans and protocols, the prospect of integrating our construct into the genome of V. natriegens was of particular interest.</p>
+ <p>Chemical compounds are able to absorb and refract light at different wavelengths depending on their structures, more precisely because of their ability to delocalise their π-electrons. The more freedom a π-electron has to move around the structure, the shorter the wavelength it can absorb, and the longer the wavelength which can be refracted. The ability of a compound to delocalize its π-electrons is determined by the consecutive overlap of multiple p-type orbitals generated by the presence of consecutive double (or triple) bonds. Compounds with an high degree of conjugation, like Beta-carotene (Fig. 1 Beta-carotene) absorb light in the blue region of the visible light spectrum (~450 nm) and refract on the opposite side of the spectrum, the red region (~750 nm), giving molecules their characteristic colours. </p>
<p>Integrating the construct into the genome would eliminate the risk of the organism dropping maintenance of the plasmid, as well as remove the need of growing it in antibiotic-supplemented media.</p>
<p>While the team was unable to reach this phase of our lab work, extensive research was carried out in order to plan the ideal method, the sum of which is listed below.</p>
`,