DNA Read, Write, & Edit

Weekly Log: Notes and autobiographical description of the progress made-

  1. The Hybrid Chronology: From Chiloé to In Silico Sprints

“Operating as a remote node in the Southern Cone, my start to HTGAA was defined by a journey even further south and a stark contrast between tradition and technology. Due to a pre-planned expedition to the Chiloé archipelago (500 km away), I prioritized family care and supporting my grandmother—a commitment that required stepping away from live connectivity.

Returning at midnight, I undertook a “post-trip night marathon,” an intensive late-night session (working until 5 a.m.) to bridge the distance. Lacking physical access to the lab and with limited time, I used AI assistants not only to summarize lessons but also as active tutors to quickly master the digital workflow. This allowed me to efficiently manage TwistDNA, SBOLCanvas, and Benchling in a single session, transforming my “Biosynthetic Bridge” concept into a validated in silico design despite the limitations of distance.”

5.1 DNA Read.

(i) What DNA would you sequence and why?

For this project, ideally, we would perform shotgun metagenomics on the sediment and water column of the discharge area in Lake Budi.

Why: We need to identify the available “hardware.” We must know exactly which native halotolerant bacteria inhabit the area to select the most robust chassis. Furthermore, we need to sequence the DNA of any fecal pathogens present to design Action 2 bacteriophages with absolute specificity, ensuring they do not harm the beneficial microbiota.

(ii) Editing Technology:

For the analysis of eDNA from mud or water samples, the Oxford Nanopore Technologies (ONT) platform will be used, a third-generation technology that allows for the real-time sequencing of single molecules. Unlike second-generation methods, ONT generates long reads, facilitating the assembly of complex genomes and the identification of species in environmental mixtures. The process begins with the extraction of environmental genomic DNA, followed by library preparation that prioritizes long fragments. After end repair, adapters are ligated with a motor protein that guides the strand into a protein nanopore embedded in an electroresistant membrane. Under a constant voltage, the passage of each nucleotide through the pore generates a characteristic disruption in the ionic current. These signal variations are processed by Recurrent Neural Network (RNN) algorithms to perform real-time base calling. Finally, the system generates FASTQ files that integrate the nucleotide sequence with their respective quality values ​​(Phred scores), allowing immediate bioinformatics analysis without the amplification biases inherent in PCR.

5.2 DNA Write:

(i) What DNA would you synthesize and why?

For the DNA writing phase, the strategy focuses on the synthesis of a Bioremediation Logic Circuit that operates as an in vivo biological computational system. This design implements a strict AND logic gate, which processes two input signals specific to the contaminated environment: the presence of skatole (a fecal marker) and hydrogen sulfide (H₂S). The genetic architecture links inducible promoters and riboswitches (e.g., [P-skat] and [riboswitch-H₂S]) in such a way that the expression of the effector gene (a cleaning hydrolase) is activated only when both contaminants are present, thus optimizing the cell’s metabolic load and avoiding energy waste in the absence of contamination. As a critical biosecurity measure, the circuit integrates a Kill-Switch module based on the toxin-antitoxin system (MazF/MazE), programming automatic cell death if the bacteria leaves the specific chemical niche, thus transforming native bacteria into intelligent, efficient, and contained synthetic agents.

(ii) Synthesis Technology: Silicon Platform

For the manufacture of genetic material, DNA synthesis on silicon chips (Twist Bioscience-type technology) will be used. This high-throughput approach scales traditional phosphoramidite chemistry (deprotection, coupling, oxidation, capping) to a massive scale. Unlike column synthesis, this method uses silicon wafers with microwells to synthesize thousands of oligonucleotides simultaneously. These oligonucleotides are then recovered and assembled using PCR or Gibson assembly to construct complete genes. While this technology allows for high production density, the experimental design must consider critical limitations such as a turnaround time of several weeks and the thermodynamic difficulty of synthesizing sequences with complex secondary structures (high GC content or repeats). This makes the synthesis of complete genomes economically unfeasible without a hierarchical assembly strategy.

5.3 DNA Edit:

(i) What DNA would you want to edit and why? The primary target for editing is the genomic DNA of native halotolerant bacteria (e.g., Halomonas sp. isolated from Lake Budi) to permanently integrate the bioremediation circuit into a chromosomal “safe harbor,” ensuring genetic stability and hereditary transmission that transient plasmid vectors cannot guarantee. Additionally, the viral genome of associated bacteriophages will be edited to enforce a strictly lytic replication cycle, effectively preventing lysogeny and ensuring the phages function solely as precise biocontrol agents that eliminate the bacterial hosts once the remediation task is complete or if containment is breached.

(ii) What technology or technologies would you use to perform these DNA edits and why? To perform these edits, the CRISPR-Cas9 system (or the Cas12a variant, optimized for high-salinity stability) will be employed, delivered as Ribonucleoprotein (RNP) complexes via electroporation to maximize precision and reduce the cytotoxicity associated with continuous plasmid expression. The protocol relies on synthetic gRNAs to guide the nuclease to specific PAM sequences, inducing a Double-Strand Break (DSB) that triggers the native Homology-Directed Repair (HDR) pathway; this mechanism utilizes the synthetic DNA module (from step 5.2) as a template to insert the circuit, a strategy chosen to mitigate off-target effects despite the challenges of low HDR efficiency in wild-type strains.

This documentation and the technical structuring were developed with the assistance of Gemini (Google AI). I used the AI as a thought-partner to synthesize lecture notes, translate technical concepts, and structure my design journey while remote.