4.1 DNA Read

(i) What DNA would you want to sequence (e.g., read) and why?

I would like to sequence the DNA of Escherichia coli (E. coli) due to its importance in biotechnology and synthetic biology. E. coli serves as a model organism for molecular biology research, and sequencing its DNA allows for better understanding of gene regulation, protein expression, and metabolic pathways. Additionally, sequencing E. coli can contribute to advancements in medical research, bioengineering, and antibiotic resistance studies.

(ii) What technology or technologies would you use to perform sequencing on your DNA and why?

I would use third-generation sequencing technologies, such as Oxford Nanopore Technology (ONT) or PacBio SMRT sequencing, due to their ability to generate long reads, which help in assembling genomes with high accuracy.

1. Is your method first-, second- or third-generation or other?
   • The selected technology belongs to third-generation sequencing as it allows direct, real-time sequencing without requiring amplification steps.
2. What is your input? How do you prepare your input?
   • Input: Genomic DNA extracted from E. coli.
   • Preparation: DNA is extracted, fragmented if necessary, and ligated with sequencing adapters. Some protocols may include PCR amplification.
3. What are the essential steps of your chosen sequencing technology?
   • DNA library preparation (extraction, adapter ligation)
   • Loading into the sequencing platform (flow cell for ONT or SMRT cell for PacBio)
   • Real-time base calling using nanopores or single-molecule fluorescence detection
   • Data analysis for genome assembly and annotation
4. What is the output of your chosen sequencing technology?
   • The output consists of raw sequence reads that are further analyzed to reconstruct the E. coli genome, identify genetic variations, and annotate functional elements.

4.2 DNA Write

(i) What DNA would you want to synthesize (e.g., write) and why?

I would synthesize a specific gene of E. coli that encodes a fluorescent protein, such as GFP (Green Fluorescent Protein). This synthesized gene could be used in genetic engineering experiments to visualize gene expression and protein localization within bacterial cells.

(ii) What technology or technologies would you use to perform this DNA synthesis and why? I would use oligonucleotide synthesis coupled with Gibson Assembly or CRISPR-based methods to ensure accuracy and efficiency in constructing the desired gene.

1. What are the essential steps of your chosen synthesis methods?
   • Oligonucleotide synthesis (chemical DNA synthesis of short fragments)
   • Assembly of synthesized fragments into the full gene sequence
   • Cloning into a plasmid vector using Gibson Assembly or restriction enzyme digestion
   • Transformation into E. coli for expression verification
2. What are the limitations of your synthesis method?
   • Length limitations in chemical synthesis (typically up to 200 nucleotides per oligonucleotide)
   • Potential errors in sequence assembly requiring verification via sequencing
   • Cost and time constraints for longer or complex sequences