Goal: Amplifying Nap A, NirS, NorB and NosZ genes to create our construct
To enhance the overexpression of our target genes in E. coli, our initial step involved the isolation of these genes, a crucial phase in our project's progression. This isolation process was executed meticulously, beginning with the design of primers tailored to the specific genes of interest. Subsequently, we embarked on the gene amplification journey, employing the Polymerase Chain Reaction (PCR) method.
It's worth noting that prior to initiating the PCR amplification, we conducted a thorough in-silico analysis, optimizing primer sequences for the intended genes. However, our actual PCR outcomes proved to be somewhat less promising than anticipated, prompting a deeper examination of our procedures.
Despite adhering to the recommended annealing temperatures provided by both SnapGene and NCBI Primer Blast, our PCR results fell short of our expectations. For instance, consider the case of the NapA gene, for which our in-silico analysis suggested an ideal annealing temperature of 57℃. Yet, in practice, the resulting PCR band exceeded our desired size, measuring larger than 10 kb, as visually depicted in Figure 1.
This discrepancy between our in-silico predictions and the experimental outcomes raised important questions and challenges that demanded our attention and further investigation to optimize our gene isolation. After conducting numerous gradient experiments and exploring alternative polymerases, we successfully determined the optimal annealing temperatures for each primer. However, we encountered a challenge where the resulting bands are not consistently robust, leading to low-concentration DNA. This limitation has hindered our ability to proceed with the subsequent step of inserting the isolated DNA into the vector.
Future Direction
Despite our thorough in-silico analysis, we've faced challenges in amplifying certain genes as expected. Our next step involves redesigning primers, particularly for the NapA gene, which encodes nitrate reductase and is pivotal in the denitrification pathway. We aim to enhance primer specificity and efficiency. Our experiences, though challenging, are valuable lessons for future iGEM teams, who can learn from our journey, including the complexities of in-silico designs versus real-world lab work. We hope to contribute insights into primer design and gene amplification for the benefit of future projects. Additionally, we'll work to optimize the entire gene amplification process, ensuring we collect sufficient genetic material for subsequent steps like restriction digests. Our commitment to improvement and sharing knowledge remains unwavering as we advance in the field of genetic engineering and synthetic biology.
Next Steps
To envision the continuation of our project, let's consider the essential steps that lie ahead. First and foremost, we need to address the challenge of gene amplification. One potential approach might involve a redesign of the primers, particularly for the NapA gene. However, it's crucial to tread carefully, as altering the primer sequence could inadvertently lead to an undesirable increase in the lengths of the flanked genes, a complication we'd prefer to avoid.
For the other three genes, we've successfully identified their ideal annealing temperatures. Yet, despite this progress, the yield of DNA remains insufficient for our requirements. To overcome this hurdle, our next course of action would involve increasing the DNA concentration. In an ideal scenario, we would implement a precipitation technique with ethanol, which holds the promise of significantly boosting our DNA yield.
Upon achieving successful gene amplification, our project could move forward as originally planned. This includes the insertion of the genes of interest into plasmids and the subsequent transformation of our E. coli BL21 strain. The ultimate goal here is to achieve a higher yield of the enzymes that are central to our research. Following the successful expression and harvesting of cells, the purification process would come into play.
In parallel to our experimental work, modeling the enzymatic activities would be a crucial aspect of our project. We would aim to model the activity of the four enzymes, each equipped with their histags. Furthermore, we would explore the activity of these enzymes when connected to a peptide linker with the supply of electrons. It would be imperative during this process to ensure that the active sites of the enzymes remain unobstructed for optimal functionality.
As we progress and set up our bioreactor, a field test with available groundwater would mark a significant milestone. This field test would provide invaluable insights into the efficiency of our bioreactor in denitrifying water under real-world conditions. Only after the successful completion of this field test could we begin contemplating the design of a scaled-up bioreactor, a prospect that holds immense potential for practical applications.
In summary, while these steps represent the envisioned progression of our project, it's important to acknowledge that we are at the stage of planning and preparation, with much work ahead of us to transform these ideas into tangible results.