<p>Each fragment has a corresponding ribosome binding site (RBS) positioned immediately upstream of the start codon to ensure proper translation. The native P1 promoter is located upstream of the gene fragments to initiate transcription. The terminator is introduced downstream of the gene sequences for proper transcription termination. Since the gBlocks have been divided into four segments, an additional set of eight primers have been designed with sequences complementary to adjacent fragments, ensuring proper alignment for Gibson assembly. Each primer adds approximately 40 base pairs of overlap, following guidelines from SnapGene, to facilitate seamless assembly of the fragments. The final recombinant plasmid has a total length of 12,413 base pairs.</p>
Figure X. 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.
Figure 8. 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 <a href="https://www.biorender.com/" target="_blank">BioRender.com</a>.
</figcaption>
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@@ -311,8 +311,37 @@ export default {
{
title:'Test',
description:`
<p>Validating the assembly results through sequencing.</p>
<p>Analyzing whether the intended constructs have been successfully created.</p>
<p>To evaluate the success of our assembly, we performed multiple tests. First, we conducted a colony PCR to get a general notion if the plasmid was integrated. Next, we miniprep the liquid cultures and conduct PCR to amplify sections of the construct, using different combinations of primers. We later would send them for sequencing using primers that were positioned inside the original backbone of pSEVA.</p>
<p><b> GA 1 & 2 </b></p>
<p>Moderate growth was observed, however, when a colony PCR was conducted with primers fragment 1 forward and 2 reverse. We then ran it on a gel electrophoresis, however the bands did not correspond to any of the expected values. We deemed it as having failed.</p>
<p><b> GA 3 & 4 </b></p>
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<img src="https://static.igem.wiki/teams/5306/engineering/ga-3-4-colony-pcr.png" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
<figcaption style="font-style: italic;">Figure 9.Colony PCR of GA 3-4</figcaption>
</figure>
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 12.Mini-prepped F3-fwd + F4-rev in better quality>
</figure>
<p>We had moderate growth on the plates again, a colony PCR was conducted again, using the same primers as above. The results proved to be inconclusive as no bands were visible. However, this time a miniprep was done from the liquid cultures. We first tested with the same primers as for the colony PCR. After checking with Snapgene, it was observed that the primers had multiple binding sites, leading to the possibility of different band lengths. We decided to test different sets of primers with fewer amounts of binding sites (Fragment 2 forward with 4 reverse, and fragment 3 forward with 4 reverse). The gel of the PCR products showed no bands at the expected lengths. We decided that the assembly had not succeeded.</p>
<p><b> GA 5 & 6 </b></p>
<p>The plates had growth, a colony PCR was conducted, however like before the bands were not at the expected lengths. We miniprepped the cultures, and sent them off for sequencing. The results of the sequencing showed that the assembly had failed.</p>
<p><b> GA 7 & 8 </b></p>
<p>No growth was observed, the construct had not assembled.</p>
<p><b> GA 9 & 10 </b></p>
<p>Growth was observed, another colony PCR was done, except this time the primers fragment 1 forward with 3 reverse, and fragment 2 forward with 4 reverse were also tested. The gel showed no conclusive bands. Minipreps were made from the liquid cultures and sent off for sequencing. After analysing the results, we saw that the assembly had failed yet again.</p>
