<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>Transformation describes the process through which bacteria are able to uptake foreign DNA into the cell. In synthetic biology, this mechanism is what allows scientists to insert plasmid constructs into cells, allowing the cells machinery to maintain and replicate the plasmid (AddGene, n.d.). Scientists have thus developed methods of inducing transformation-readiness in cells (referred to as competence), with various strains modified for ease of transformation and improved plasmid maintenance.</p>
<p>In the process of literature review and protocol preparation, three primary methods of transformation were found and put under consideration. The third of these two, the preparation of naturally competent cells, was ruled out due to time constraints and the difficulty of the preparation, requiring the overexpression of a tFoX gene (See Module “Genomic Integration” for more information).</p>
<p>This left two options; chemical (heat shock) transformation, or electroporation transformation.</p>
<p>Electroporation transformation was suggested by Weinstock et al. (2016) to have a higher transformation efficiency than heat shock, with Tschirhart et al. (2019) confirming it’s effectiveness in transforming assemblies in the pSEVA261 plasmid. However, due to the team lacking access to the necessary equipment for the carrying out of electroporation, and insufficient funding or time to acquire or construct equipment, the decision was made to use heat shock transformation.</p>
<p>Heat shock transformation, while outlined in Weinstock et al. (2016) as having a lower transformation efficiency, was still proven to work well.</p>
<p>Chemically competent cells were prepared in accordance with previously compiled and adjusted protocols. As we were working with a novel chassis, V. natriegens, competent cells were not available for purchase the way that E. coli cells are. This protocol consists of soaking the cells in a cation solution, which enhances the cell walls permeability, followed by rapid freezing at -80 °C via a liquid nitrogen bath and storage at -80 °C until use.</p>
<p>The heat shock protocol makes use of the membrane permeability of the cells, with plasmid DNA being added to thawed competent cells. This mixture is incubated on ice for 30 minutes, followed by exposing the cells to 42 °C heat for 45 seconds and incubation at 30 °C for 2 hours. These steps prompt the cell to uptake the plasmid DNA, in thanks to the cell wall permeability.</p>
`
},
{
title:'Build',
description:`
<p>Final assembly of the constructs.</p>
<p>Using all accumulated knowledge to achieve the best results.</p>
<p>The protocols for the preparation of chemically competent cells and the heat shock transformation itself were sourced from Weinstock et al. (2016) and Tschirhart et al. (2019), and adapted for the equipment available.</p>
<p>No significant changes to the volumes, temperatures, or suggested conditions were applied in the transformation attempts carried out.</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>To check the viability of our chemically competent cells, an empty pET39b backbone was transformed into the Vibrio natriegens. This proved to be a success as growth was observed.</p>
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<img src="https://static.igem.wiki/teams/5306/engineering/pet39b-plasmid-in-v-nat.jpg" alt="Colony PCR of GA 3-4" style="width:100%; height:auto;" />
<figcaption style="font-style: italic;">Figure 18.xPlates containing colonies of V. natriegens transformed with pET39b. </figcaption>
</figure>
<p>It was then replicated with five samples; an empty pSEVA261 plasmid and four of the full constructs. However, no colony growth was observed, leading the team to draw conclusions and re-calibrate our approach.</p>
`
},
{
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>Discussion following each of the failed transformation allowed us to draw conclusions and eliminate possible causes of the unsuccessful transformations.</p>
<p>Previous assembly and transformation attempts in pET39b yielded colonies, proving there was no issue with the chemically competent Vibrio natriegens.</p>
<p>While previous transformations into V. natriegens yielding no colonies, this was thought to be due to the Gibson Assemblies not working as intended, resulting in a lack of kanamycin resistance in the transformed cells. However, with the transformation of the fully assembled construct, the culturing conditions (incubation at room temperature vs. 37 °C), and the media (plates containing kanamycin and 3% w/v NaCl) can be ruled out as causes. Additionally, a lack of colonies on the empty pSEVA261 sample plate rules out the cause being the fully assembled construct as well.</p>
<p>Evidence points to the plasmid pSEVA261 being responsible for the absence of colonies, possibly due to plasmid incompatibility with the chassis.</p>
<p>We aim to test this hypothesis by carrying out assembly and transformation attempts in the pET39b, both of single genes and the fully assembled construct, in order to verify the exact cause of the absence of transformed colonies with pSEVA.</p>
<p>We will however at the same time transform the construct and pSEVA backbone, adapting the heat shock transformation.</p>
`
},
]
...
...
@@ -513,7 +529,7 @@ export default {
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 17. Beta-carotene. Image sourced from Wikipedia</figcaption>
<figcaption style="font-style: italic;">Figure 19. 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>
...
...
@@ -521,19 +537,19 @@ export default {
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 18. Griess test reaction scheme. Image sourced from Wikipedia</figcaption>
<figcaption style="font-style: italic;">Figure 20. 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;">
<figcaption style="font-style: italic;">Figure 19. Indophenol blue reaction scheme. Image sourced from Sasongko, A. (2018).</figcaption>
<figcaption style="font-style: italic;">Figure 21. 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;">
<figcaption style="font-style: italic;">Figure 21: pJUMP21-1A plasmid. Sourced from SnapGene.</figcaption>
<figcaption style="font-style: italic;">Figure 23: pJUMP21-1A plasmid. Sourced from SnapGene.</figcaption>
</figure>
<p>The overexpression of tFoX gene and induction of natural competence for genome integration was also considered, with Dalia et al. (2017) and Specht et al. (2024) both outlining the possibilities this route opens up. However, this option was discarded as it was considered beyond our reach to carry out engineering on V. natriegens to induce natural competence prior to having a completed assembly without integration in V. natriegens, considering the factor of the time available to us.</p>
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 22. A comparison of the Natural Competence Transformation workflow (A) made possible via tFoX gene overexpression, and the standard Heat Shock and Electroporation workflows (B). Sourced from Specht et al. (2024)</figcaption>
<figcaption style="font-style: italic;">Figure 24. A comparison of the Natural Competence Transformation workflow (A) made possible via tFoX gene overexpression, and the standard Heat Shock and Electroporation workflows (B). Sourced from Specht et al. (2024)</figcaption>
</figure>
<p>The large size of the insert shifted the focus to serine integrases since it was shown that these enzymes can successfully insert up to 10k bases of inserts during a reaction, with high efficiency, unidirectionally and no need for additional proteins (Snoeck et al., 2019). Serine integrases work by recognising attachment sites on the plasmid and the genome of the host, attB and attP, which tend to be around 50bp of nucleotides flanking the area of interest (Merrick et al., 2018; Muroi et al., 2012). The small size of the att sites make them easier to insert via homologous recombination, making it possible to use integrases that are not native to the chosen chassis.</p>
<!-- Image with figure caption -->
<figure style="text-align: center; margin: 20px 0;">
<figcaption style="font-style: italic;">Figure 23. Image illustrating the working mechanism of integrases, taken from Merrick et al., 2018. </figcaption>
<figcaption style="font-style: italic;">Figure 25. Image illustrating the working mechanism of integrases, taken from Merrick et al., 2018. </figcaption>
</figure>
<p>With this background research done the team planned to use homologous recombination to insert the phiC31 att sites into the genome of V. natriegens making them compatible for the use of phiC31 integrase (Merrick et al., 2018). The location of the insertion sites were chosen based on the research done by a previous iGEM team VibriGens since they have already identified suitable regions with low transcription numbers, non-coding areas and 500bp of space between coding sequences (Marburg, 2018). Followed by the integration of a GFP gene to be able to easily track and test the success of the integration. The desired operon would have been inserted only after the location and presence of the att sites were confirmed. However, due to time and financial constraints the team was not able to carry out this phase of the planned research, and instead focused on successfully assembling the traditional plasmid and transforming the V. natriegens.</p>
<p>Even if we were not able to carry out this research ourselves, we are hoping that this information will prove to be useful for future iGEM or research teams. </p>
