Week 2 HW: DNA read, write and edit

Part 1: Benchling & In-silico Gel Art

In order to create my gel art, I first created an account in Benchling and signed into it. Then, I found and downloaded the genebank of the Lambda DNA from New England Biolabs. After that, I simply pasted the DNA sequence into my Benchling workspace, and started experimenting with the different digestions possible using the allowed enzymes.

Eventually, I came to a design I believed to be symmetrical and graphically pleasing to look at. A nice, bored looking elephant!

The design itself

What I had in mind, image created by me via Pixil

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

Unfortunetly, I did not have he required lab access to complete this part. Hopefully, I could come back and revisit it once I do.

Part 3: DNA Design Challenge

3.1. For my protein, I chose the ardA anti restriction protein, found in many strains of E. coli. My reason for choosing this protein is related to one of my original ideas for my final project. Which was to incase the phages used to eliminate antibiotic resistent bacteria, such as Salmonella enterica, with the proteins that block the restriction enzymes that protect the bacteria from these viruses. So, I found and downloaded the sequence of my protein in Unipprot.

3.2 & 3.3. Afterwards I reverse translated my protein sequence in Cusabio, and codon optimized it using Vector Builder.

Reverse translation using Cusabio

Codon optimization using Vectro Builder

3.4. For the production of my protien, I belive In vitro, or cell-free protein expression to be an optimal technique. As although in vitro expression is not practical for commercial large-scale recombinant protein production, it has a variety of features that make it considerably more useful and flexible for many research applications, such as labeling of proteins with stable isotopes for structural analysis and production of functional virons or toxic polypeptides.

Part 4: Prepare a Twist DNA Synthesis Order

I had a lot of trouble sigining into Twist due to the fact that I am not part of an institution or lab that. As mentioned before, I am in highschool, and was not sure if that really could be counted. Adding to that problem is the fact that my VPNs, which I need to use Twist, have been acting up. Hopefully I can get them running until the next lecture.

Part 5: DNA Read/Write/Edit

5.1. i. If given no limitation, I would want to read and sequnce the DNA of the naked mole rat. This creature is virtually immune to cancer and many other age-related decline, therefore managing to somehow, someday, impliment its secrets into humans in a safe and ethical way could be a wonderful advance in biology. ii. 1. To sequence the Naked Mole Rat (NMR), I would choose Pacific Biosciences (PacBio) HiFi Sequencing. Specifically, I would use the PacBio Revio system. While standard short-read sequencing (like Illumina) is great for basic spell-checking, the NMR’s “superpowers” likely lie in complex structural variations—gene duplications, insertions, and repetitive regions—that short reads simply cannot resolve. To see the full picture, we need long, unbroken reads with high accuracy. This method is classified as Third-Generation Sequencing. Third-generation (PacBio, Nanopore) sequences single molecules of DNA in real-time without the need for PCR amplification to generate the signal. It observes the DNA polymerase adding nucleotides as it happens. 2. The input is High Molecular Weight (HMW) Genomic DNA, and its essential steps are: 1- Extraction: We extract gDNA using a gentle lysis method to keep fragments massive. 2- Shearing: We use mechanical shearing (like a Megaruptor) to break the DNA into a consistent, large size range. 3- DNA Damage Repair: Enzymes are added to fix any nicks or gaps in the sugar-phosphate backbone caused by the shearing. 4- Adapter Ligation (The Critical Step): We ligate SMRTbell adapters to both ends of the double-stranded DNA. 5- Primer Annealing & Polymerase Binding: We anneal a sequencing primer to the adapter and bind the DNA polymerase enzyme to the complex. The sample is now ready for the machine. 3. The primary output is HiFi Reads.Format: Typically FASTQ or uBAM files.Characteristics:Length: Average read lengths of 15kb to 20kb (kilobases).Accuracy: Q30 or higher (99.9% accuracy).Value: This output provides the best of both worlds: the length to span difficult repetitive regions (common in complex mammal genomes) and the accuracy to detect tiny mutations (SNPs) without needing massive computational correction.

5.2. i. If, once again, given not limits, I would synthesize and refactor the entire Nitrogen Fixation (nif) gene cluster (approx. 20 genes) found in soil bacteria, optimized for expression in cereal crops like corn or wheat. This is arguably the most impactful application for global sustainability. It would eliminate the need for synthetic nitrogen fertilizers, which are a massive source of greenhouse gases and water pollution (runoff). It would trigger a second Green Revolution, allowing crops to “feed themselves.” ii. To synthesize the genetic system I described, I would use Enzymatic DNA Synthesis (EDS). 1. Enzymatic synthesis builds DNA one base at a time using a “Stop-and-Go” cycle. Firstly, A starting DNA fragment (an “initiator”) is tethered to a solid support surface in a microscopic well. Then, An engineered TdT enzyme and a specific nucleotide (A, T, C, or G) are added. TdT is a “template-independent” polymerase, meaning it can add bases without needing a guide strand. After that The excess enzymes and unused nucleotides are washed away, and mild solution is added to remove the “blocker” (the terminator) from the newly added base. This exposes the “hook” (the 3’-OH group) so the next base can be attached. 2. While EDS is the “frontier” technology of 2026, it still faces hurdles in three key areas, such as speed, accuracy, and scalability.

5.3. i. Lastly, I would edit the TP53 gene in human somatic cells, often called the “Guardian of the Genome”. In humans, we have two copies of p53. If they mutate, cancer often follows. I would edit the genome to include multiple redundant, hyper-sensitive copies of this gene, similar to what we see in elephants (who rarely get cancer despite their size). This would effectively “auto-update” our cellular security system. The moment a cell starts to turn cancerous due to UV damage or chemical exposure, the enhanced p53 would detect the break and force the cell to repair itself or safely self-destruct. ii. To perform the targeted DNA edits described, I would use a combination of CRISPR-Cas9, Base Editing, and Prime Editing. 1. The Cas9 enzyme creates a Double-Strand Break (DSB) at a precise location. The cell then tries to fix this break using Non-Homologous End Joining (NHEJ). This process is messy and often deletes or inserts random bases, which effectively “breaks” or silences the gene. These use a “deactivated” Cas9 (dCas9 or nickase) that cannot cut both strands of DNA. Instead, it is fused to other enzymes. 2. Editing requires meticulous “software design” before the biological work begins. First, we’ll identify the exact genomic coordinates. Then, we use software to create a 20-nucleotide sequence that matches the target. We must ensure a PAM sequence exists next to our target. 3. A major limitation is Mosaicism. Even with a near perfect edit, the technology might only work in about maybe 70% of the cells in a sample.