Homework #2 Questions (scroll down for answers)

  1. DNA Read
    1. 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 want
    2. What technology or technologies would you use to perform this sequencing and why? From 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?
  2. DNA Write
    1. 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 bio materials (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! :)
    2. What technology or technologies would you use to perform this DNA synthesis and why?
  3. DNA Edit
    1. What DNA would you want to edit and why? In class George shared a variety of ways to edit the genes and genomes of humans and other organisms. Such DNA editing technologies have profound implications on human health, development, and even human longevity and human augmentation. What kinds of edits might you want to make to DNA (e.g., human genomes and beyond) and why?
    2. What technology or technologies would you use to perform these DNA edits and why?

Homework #2 Answers and Responses

  1. DNA Read

    1. I want to sequence the ammonium oxidation pathway and associated genes in the globally distributed marine Thaumarchaeota. While this has been sequenced before in this organism multiple times over, I am interested in surveying the diversity of genes and pathways related to the system in order to understand the nature flexibility of this system and the environmental ranges upon which it operates.
    2. I would and am using Illumina sequencing for this exact tasks, but there are strong arguments for leveraging a technology like PacBio or Oxford Nanopore to ensure longer more contiguous assembly of fragments.

  2. DNA Write

    1. I would like to synthesize the amoA subunit (and express the protein along with it’s required subunits) because this is the supposed active site of the multi-complex ammonium monooxygenase enzyme. I will refrain from sharing the exact sequence because I don’t know how to pick a single one to synthesize (this is a big part of the problem!)
    2. Gibson assembly seems to be the superior method in this cases because it allows for synthesis of genes >100 nucleotides in length, which includes the amoA gene.

  3. DNA Edit

    1. I would be interested in engineering genes to enhance human metabolism through enhanced enzyme efficiency. Ultimately all food-based energy is some finite quantity, so theoretically, if our enzymatic machinery was made to be more efficiently then our food requirements could decrease. This would have many impacts including on global poverty and food supply in the developing world.
    2. CRISPR-Cas based genome editing, because of it’s accuracy and precision, would be most ideal for this use case. However, I will admit a lack of expertise on this topic and so an alternative toolkit might be more suitable and appropriate.

    Gel Art (from Benchling interactive notebook)

    Description: I discovered a deep-subseafloor genome representing the first sequenced organism of its phyla. As an honorific designation, one of the strains was subsequently named after me (Candidatus Pyrohabitans jungbluthii) (Reference: https://www.science.org/doi/10.1126/sciadv.abm9651). First, I digested that genome, for funzies, along side Lambda DNA with standard restriction enzymes.

    virtual_digest_sequence_Lambda_NEB.png

    For something more refined, I did away with the archaeal genomic DNA because it is a blur. Instead I used the Lambda DNA and focused on showing bands with 20, 19, 18, …, 2, 1 restrictions total, just to see a pattern and get a feel for the range of restriction enzyme options. This is the result, for which notably some integer values are missing because of a lack of an enayme making that specified number of cuts.

    virtual_digest_sequence_Lambda_NEB (1).png

    It strikes me as a bit of a Rorschach test, but I kind of see the United States coastline shaping up on the right hand side. The left side requires some work…

    <ominous sounds of power tools>

    Third attempt!

    virtual_digest_sequence_Lambda_NEB (2).png

    The outline of the southern US border is perhaps a bit better but the rest leaves a lot to be desired. Florida didn’t turn out as anticipated, easy to fix that by removing a segment. I also tried duplicate digests in side-by-side lanes (#3 and #4) but don’t like how it turned out, will remove one.

    Fourth attempt!

    virtual_digest_sequence_Lambda_NEB (3).png

    Better, but again, as someone who likes maps, this leaves a lot to be desired. May have to call it there, however, since this has been a useful proof of concept showing what is possible with Benchling.