Steven Ness

(1) DNA Read.

(i) What DNA would you want to sequence (e.g., read) and why? This could be DNA related to human health (e.g. genes related to disease research), environmental monitoring (e.g., sewage waste water, biodiversity analysis), and beyond (e.g. DNA data storage).

I would read the DNA from different species of the family Muricidae, these sea snails are the source of the ancient dye, Tyrian Purple. These sea snails, known as murex in ancient times have a gland that produces 6,6'-dibromoindigo in very small quantities, approximately ten thousand murex must be sacrificied to produce a gram of 6,6'-dibromoindigo.

I would sequence the DNA and the mRNA in the color producing gland of one species of murex and would attempt to find out what is the biochemical pathway that produces 6,6'-dibromoindigo. I would then sequence a number of related species in the family Muricidae to find if there are interesting or useful mutations that could allow us to change the specificity of the enzymes to produce other compounds like 6,6'-dichloroindio or 6,6'-diiodoindigo.

https://en.wikipedia.org/wiki/Muricidae

(ii) What technology or technologies would you use to perform this sequencing and why? In lecture, a variety of sequencing technologies were mentioned. Which one(s) do you think would be most suitable for the DNA you want to read and why?

I would use the Illumina Next Generation Sequencing (NGS) platform to sequence these genomes. It is similar to Capiliary Electrophoresis (CE) sequencing in that DNA polymerase is used to catalyze the incorporation of fluorescently labled deoxyribonucleotide triphosphates (dNTPs) into a DNA template strand during cycles of DNA synthesis. During each cycle of this process, a nucleotide is incorporated and is identified by the excitation of an attached fluorophore. The big difference is that while CE sequences one DNA fragment, NGS extends this process across millions of fragments in a massively parallel architecture.

At its heart it uses Sequencing By Synthesis (SBS) chemistry, where the sequencing procedure is carried out by performing a synthesis reaction. This is one of the most common ways to do sequencing currently.

To sequence the mRNA we would use a reverse transcriptase to copy the mRNA back to cDNA and then sequence the cDNA. This would give a picture of the transcriptome of the cell, to find which proteins were being expressed.

https://www.illumina.com/content/dam/illumina-marketing/documents/products/illumina_sequencing_introduction.pdf

(2) DNA Write.

(i) What DNA would you want to synthesize (e.g., write) and why? These could be individual genes, clusters of genes or genetic circuits, whole genomes, and beyond. As described in class thus far, applications could range from therapeutics and drug discovery (e.g., mRNA vaccines and therapies) to novel biomaterials (e.g. structural proteins), to sensors (e.g., genetic circuits for sensing and responding to inflammation, environmental stimuli, etc.). If possible, include the specific genetic sequence(s) of what you would like to synthesize! You will have the opportunity to actually have Twist synthesize these DNA constructs! :)

I would write a set of genes to synthesize Tyrian Purple, 6,6'-dibromoindigo in E. coli. With these genes, I would grow large quantities of E. coli and would produce this chemical.

A paper from 2021 describes one approach to this problem, and they report success by using a combination of two different enzymes. The product of the first enzyme interferred with the reaction of the second enzyme, so they found that they needed to spatio-temporally separate the processes to achieve good yields of 6,6'-dibromoindigo.

After replicating this experiement, I would then try to further optimize the enzymes and their reactions using methods from computational chemistry including Alphafold2 and the Gnina docking software. I would try to find a way to combine the enzymes to make this a single step procedure or to optimize it in other ways.

https://www.nature.com/articles/s41589-020-00684-4#:~:text=Direct fabric dyeing using E,mixed for further oxidation12

(ii) What technology or technologies would you use to perform this DNA synthesis and why?

I would used a Next Generation (Chip Based) DNA synthesis method where the DNA synthesis is minaturized and put onto a integrated chip like substrate in a way similar to how Twist works.

This method uses conventional chemistry for DNA synthesis, but instead of doing this chemistry in a 96 well plate, performs it on a 9600 well silicon wafer of approximately the same size.