From 4695c53580e244d89650f77fb1a5a7de6f0a3d5b Mon Sep 17 00:00:00 2001
From: Devyani Ravi <devyaniravi2003@gmail.com>
Date: Sun, 29 Sep 2024 22:21:31 +0000
Subject: [PATCH] cycle 3
---
docs/.vuepress/components/IterativeCycle.vue | 39 +++++++++++++++-----
1 file changed, 30 insertions(+), 9 deletions(-)
diff --git a/docs/.vuepress/components/IterativeCycle.vue b/docs/.vuepress/components/IterativeCycle.vue
index 3694445..7820ff3 100644
--- a/docs/.vuepress/components/IterativeCycle.vue
+++ b/docs/.vuepress/components/IterativeCycle.vue
@@ -407,34 +407,55 @@ export default {
]
},
{
- title: 'Cycle 3: Finalization',
+ title: 'Cycle 3: Successful Construct (GA 15)',
phases: [
{
title: 'Design',
description: `
- <p>Finalizing designs for the constructs to be used in experiments.</p>
- <p>This step includes ensuring all parts are ready for testing.</p>
+ <p><b> TOPO TA Cloning kit </b></p>
+ <p>To amplify the g-blocks the TOPO® TA Cloning® Kit was used. It works by introducing the fragment to a plasmid containing thymidine overhangs and covalently bound Topoisomerase I. This means the fragments need an adenine overhang to bind to the plasmid. The plasmid is then transformed into the TOPO E. coli cells, which are then plated. As the fragment is now introduced into a plasmid, as colonies grow the fragment gets amplified. When liquid cultures are made, and then mini-prepped, it is possible to perform a PCR to cut out the fragments. This should lead to less damage to the fragments and a larger concentration. The mini preps may also be sent for sequencing to see the integrity of the fragments.</p>
+ <p><b> Impact on Construct </b></p>
+ <p>We assumed the issues we were running into was due to the amplification of the g-blocks using PCR so we went back to building the full construct, with the four fragments. This meant that the pathway would remain the same, transporting and converting the nitrates to proteins.</p>
`
},
{
title: 'Build',
description: `
- <p>Final assembly of the constructs.</p>
- <p>Using all accumulated knowledge to achieve the best results.</p>
+ <p>We integrated the fragments into the TOPO plasmids, by first adding Taq polymerase to the original g-blocks. They were then all transformed into the TOPO cells, and then plated. Growth was only observed in TOPO cells with fragments 3 and 4, these cells were miniprepped and sent for sequencing. As the sequencing results showed the full fragments, they were cut out from the TOPO plasmid with PCR. As no growth was observed from TOPO cells with fragments 1 and 2, we decided for the next Gibson attempt (GA 15) we would use the original g-blocks for those two fragments. In the Gibson mixture, the linearised pSEVA, Fragments 3 and 4 were all diluted to match the concentration of fragments 1 and 2. We followed a 1:1 molar ratio of vector to fragment. Then it was transformed into NEB 10-beta E. coli competent cells.</p>
`
},
{
title: 'Test',
description: `
- <p>Conducting final tests on the assembled constructs.</p>
- <p>Verifying that the constructs perform as intended in biological systems.</p>
+ <p>The plates had significantly more growth than previous plates (25 colonies per plate compared to the 10 colonies previously seen). Colony PCR was conducted with primers fragment 2 forward and fragment 3 reverse, and 20 liquid cultures were made in total. The colony PCR results showed none of the expected bands. We decided to use a different method to test, as colony PCR had not been successful in our previous attempts. All the liquid cultures were mini-prepped and sent off for sequencing. The sequencing results of all the minipreps only sequenced Fragment 4 yet indicated that we had the full construct, however, gaps and mismatches occurred in certain constructs. Unconfident in our sequencing primers, we conducted a restriction digestion test using XhoI and NaeI restriction enzymes from NEB on four of the 20 minipreps. We created a simulation of this restriction digestion via SnapGene, which provided us with a comparative guide. The simulated pattern showed the ideal digestion pattern for the correct construct sequence. The ideal bands are seen in columns 1 and 3. When comparing the simulation and the gel, we observed bands at 12 kbp, corresponding to the full construct, in all samples. In the first, multiple additional ideal bands were created by the restriction enzymes around 7 kbp, 3 kbp, and 1.6 kbp. The presence of the full assembly fragment band size, as well as the expected digestion pattern, indicates the successful assembly of the construct. Additionally, we did not find any 2.3 kbp bands, which correspond to the pSEVA261 plasmid, indicating that there were only assembled plasmids. </p>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/whatsapp-image-2024-09-23-at-4-10-15-pm.jpeg" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 14.Snapgene simulation of restriction enzyme digestion.</figcaption>
+ </figure>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/ga-15-redigestion-set-1.png" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
+ <figcaption style="Figure 15.Results of first restriction. /figcaption>
+ </figure>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/8f608f7a-58e8-476f-b8d1-306647e9d175.jpg" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 16.Results of first restriction with better visualisation.</figcaption>
+ </figure>
+ <!-- Image with figure caption -->
+ <figure style="text-align: center; margin: 20px 0;">
+ <img src="https://static.igem.wiki/teams/5306/engineering/ga-15-redigestio-set-2.png" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
+ <figcaption style="font-style: italic;">Figure 17.Results of second restriction. </figcaption>
+ </figure>
+
`
},
{
title: 'Learn',
description: `
- <p>Gathering all data and insights from the final testing phase.</p>
- <p>Planning next steps based on the outcomes of the assembly process.</p>
+ <p>As we had a successful assembly, we came to the conclusion that the previous attempts had failed because of the amplified fragments. We suspect that the PCR and primers were altering the fragments and not allowing the g-blocks to bind to each other to form the construct. We found that the TOPO cloning kit was the most successful method to amplify our g-blocks and that working with the original g-blocks was also successful if we could get a high enough concentration.</p>
+ <p>We learnt that when assembling a construct with multiple inserts, it's important to keep a stock of higher-concentration g-blocks, provided they already contain the necessary overhangs. We also learnt the value of asking for help from multiple sources. This introduced us to new methods, such as using TOPO cells, which greatly contributed to our success. Lastly, persistence was key, despite it taking 15 attempts, we eventually succeeded in building the construct.</p>
`
},
]
--
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