<p>The fragments were amplified again, however, this time only 10 cycles were used. We also made duplicates with the standard 30 cycles. For the annealing temperature, we started using the melting point of primers (subtracting 5°C), as it did not effect the quality and yield of fragments. GA 5a and 6a, used fragments from the 10 cycles, while the GA 5b and 6b used fragments from the 30 cycles. pSEVA was linearised and had a DpnI cleanup. The Gibson Assembly ratio, vector to fragment, was also modified, GA 6a and 6b had a ratio of 1:2, while we kept the ratio for GA 5a and 5b of 1:1. We replaced the milliQ water with nuclease free water, this was used for the rest of the experiments. For transformation, GA 5a and 6a, 2μL of the assembly was introduced to the competent cells, while GA 5b and 6b, 5μL was introduced into the cells.</p>
<p><b> GA 7 & 8 </b></p>
<p>In this attempt, the original g-blocks were used, however, they had a concentration of 10 ng/μL, which meant that either the pSEVA would need to be diluted, or a very small amount used. Both were tested, using a 1:1 ratio of vector to fragment. As can be seen in table X. GA 7 used 0.2 μL, but a higher concentration, while in GA 8, it was diluted to 100 ng/μL. The Gibson Assembly Master Mix was changed to a NEBuilder®HiFi DNA Assembly Master Mix, this was used for the rest of the attempts. Each GA was transformed into two cells, with different elevated volumes than the recommended as they had low concentrations. The first set of cells received 6.4 μL, while the second set used 13 μL.</p>
<p>In this attempt, the original g-blocks were used, however, they had a concentration of 10 ng/μL, which meant that either the pSEVA would need to be diluted, or a very small amount used. Both were tested, using a 1:1 ratio of vector to fragment. As can be seen in table X. GA 7 used 0.2 μL, but a higher concentration, while in GA 8, it was diluted to 100 ng/μL. The Gibson Assembly Master Mix was changed to a NEBuilder®HiFi DNA Assembly Master Mix, this was used for the rest of the attempts. Each GA was transformed into two cells, with different elevated volumes than the recommended as they had low concentrations. The first set of cells received 6.4 μL, while the second set used 13 μL.</p>
<p><strong>Gibson Assembly 7</strong></p>
<table>
<thead>
...
...
@@ -365,34 +365,43 @@ export default {
]
},
{
title:'Cycle 2: Optimization',
title:'Cycle 2: Two Insert Construct (GA 11-14)',
phases:[
{
title:'Design',
description:`
<p>Reassessing and improving the design of DNA fragments.</p>
<p>Ensuring sequences are optimized for assembly efficiency.</p>
<p>In this phase, we reconsidered our approach and focused on inserting only two key fragments: Fragment 1 and Fragment 2. The successful integration of these fragments would result in the complete transporter system encoded by nasFED, which is essential for the uptake of NO₃⁻. Additionally, a part of Nas, namely the small subunit of the enzyme, would also be functional.</p>
<p>This construct retains the key elements necessary for effective uptake of NO₃⁻ from the environment, while its size is significantly reduced to 6,813 base pairs, as compared to the initial construct of 12,413 base pairs. This reduction in the size of the construct simplifies the overall cloning process by decreasing the complexity of the assembly, as there are fewer components that need to be ligated. Additionally, the stability of the expressed construct may be enhanced.</p>
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 13.Construct containing two fragments>
</figure>
`
},
{
title:'Build',
description:`
<p>Executing the optimized assembly protocols.</p>
<p>Combining DNA fragments with refined conditions to enhance yield.</p>
<p><b> GA 11 & 12 </b></p>
<p>As we suspected that the fragments were being damaged by the PCR, we decided to alter the annealing temperature following Thermo Fisher primer melting point calculator. The annealing temperature were changed from 55.5 to 70.5 °C for fragments 1 and 2 and changed to 70 °C for pSEVA. The 10 cycles were used on the fragments, and 30 for pSEVA. Gibson Assembly recommends when inserting 2 fragments, changing the ratio to 1:2, and an incubation time of 15 minutes. We decided to change the competent cells from 10-beta cells to NEB® 5-alpha Competent E. coli (High Efficiency). They still have a high transformation efficiency, however, they are less viable with larger constructs, but as we had a smaller construct, this was not a problem. They were also transformed into our Vibrio natriegens chemically competent cells. GA 11 and 12 had the same setup, except we changed the linearised plasmid between.</p>
<p><b> GA 13 & 14 </b></p>
<p>GA 13 and 14 were conducted at the same time as GA 11 and 12, the only factor that changed was the incubation time for the GA, instead of 15 minutes it was changed to 30 minutes. GA 13 used the same linearised pSEVA as GA 11 and GA 14 used the same linearised pSEVA as GA 12. They were only transformed into the Vibrio natriegens competent cells.</p>
`
},
{
title:'Test',
description:`
<p>Testing the results of the optimized assembly.</p>
<p>Performing sequencing and functional assays to ensure success.</p>
<p><b> GA 11 - 14 </b></p>
<p>None of the cultures grew, meaning that they were all unsuccessful.</p>
`
},
{
title:'Learn',
description:`
<p>Documenting lessons learned from optimization tests.</p>
<p>Using this information to inform future cycles of assembly.</p>
<p><b> GA 11 - 14 </b></p>
<p>As we had changed many parts between the previous attempts, we cannot pinpoint what could have led to the unsuccessful assemblies. We believed it was a mix of elements, the higher annealing temperatures could have led to more point mutations, and affected the primer binding sites in the fragments (Garibyan & Avashia, 2013). Using different cells for transformation might have affected it as well, as we had not yet tested if our V. natriegens cells were chemically competent. These tests still proved to be useful as we now understood the source of the problem. We feared that the size was too large, however, even when the size was halved it did not lead to any assemblies. We concluded that the source of the problem was the amplification of the g-block. The PCR was altering the fragments, not allowing them to form the final construct.</p>
<p><b> Next Steps </b></p>
<p>We needed to amplify the fragments without damaging them. We were guided to use TOPO cells as it would amplify the g-blocks and we would be able to sequence and check the integrity of the fragments. Allowing to have a high concentration of fragments and plasmid for the Gibson Assembly.</p>
