First, describe a biological engineering application or tool you want to develop and why. Engineered bacteriophage as a delivery vector or indicator. Factors such as selective host range, ability to integrate, infection of drug-resistant bacteria, reproducibility, and more would make them a versatile tool.
Next, describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals. (slightly edited from example framework)
Part 1: Benchling & In-silico Gel Art My node (W&M) is using one of our phages’(Kampy) DNA for the Gel Art
DNA - https://phagesdb.org/phages/Kampy/ Our lab’s restriction enzymes: AfIII Age1 AluI ApaI BamHI-HF BbsI BsaI-HF BglI BstXI DpnI Eco0109I Eco47III EcoICRI EcoRI-HF EcoRV HindIII I-SceI KpnI NotI-HF PspAI PstI-HF SpaI-HF XbaI XhoI Part 2: Gel Art - Restriction Digests and Gel Electrophoresis Our design
Python Script for Opentrons Artwork Designs Using the GUI at opentrons-art.rcdonovan.com
Using custom design tools (trying out stippling, different dot sizes)
Python Files What the different dot design looks like after programming so far (still need to scale a bit)
Subsections of Homework
Week 1 HW: Principles and Practices
First, describe a biological engineering application or tool you want to develop and why.
Engineered bacteriophage as a delivery vector or indicator. Factors such as selective host range, ability to integrate, infection of drug-resistant bacteria, reproducibility, and more would make them a versatile tool.
Next, describe one or more governance/policy goals related to ensuring that this application or tool contributes to an “ethical” future, like ensuring non-malfeasance (preventing harm). Break big goals down into two or more specific sub-goals.
(slightly edited from example framework)
Enhance Biosecurity
• By preventing incidents
• By helping respond
Foster Lab Safety
• By preventing incidents
• By helping respond
Protect the environment
• By preventing incidents
• By helping respond
Ensure Equitable Use
• Regulation
• Education
Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).
Considering actors like researchers, industry/corporations, governments and health organizations, manufacturers, and users/general public.
Goals include:
• Education (best practices, use, risks, etc.)
• Guidelines (patenting, manufacturing, use cases, who can buy, development, etc.)
Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals:
Does the option:
Education
Guidelines
Monitoring
Enhance Biosecurity
• By preventing incidents
2
1
3
• By helping respond
3
2
1
Foster Lab Safety
• By preventing incident
1
2
n/a
• By helping respond
n/a
2
1
Protect the environment
• By preventing incidents
2
1
n/a
• By helping respond
3
2
1
Ensure Equitable Use
• By preventing incidents
2
1
n/a
• By helping respond
3
1
2
Last, drawing upon this scoring, describe which governance option, or combination of options, you would prioritize, and why. Outline any trade-offs you considered as well as assumptions and uncertainties.
I would prioritize guidelines for industry and manufacturing actors, education for the general public/users and for researchers, and monitoring by governments and health agencies. Preventative measures (guidelines/education) are important to preventing misuse, unequal use, or dangerous impacts, while monitoring and having response plans are important for responding to problems. There needs to be a balance of governance for all actors, with most of my three categories being utilized for each group in different ways.
Reflecting on what you learned and did in class this week, outline any ethical concerns that arose, especially any that were new to you. Then propose any governance actions you think might be appropriate to address those issues.
Ethical concerns surround the use and possible misuse of synthetic biology. Governance actions in a lot of emerging areas are seen on the research side and health agency sides, but I believe that as use becomes broader, it will be important to have regulation for industry and education for the general public (for example, a lot of people have never heard of phage therapy).
Assignment (Week 2 Lecture Prep)
Homework Questions from Professor Jacobson:
Nature’s machinery for copying DNA is called polymerase. What is the error rate of polymerase? How does this compare to the
length of the human genome. How does biology deal with that discrepancy?
Error rate is 1:10^(6) while the human genome is about 3.2 Gbp. Biology deals with this through error checks and correction methods, including proof reading, redundancy, and cell self-destruction.
How many different ways are there to code (DNA nucleotide code) for an average human protein? In practice what are some of the
reasons that all of these different codes don’t work to code for the protein of interest?
There are many different ways to code for the same protein due to multiple codons leading to the same amino acid (redundancy). In practice, all of those different codes don’t work or aren’t seen creating the protein due to base pair and tRNA bias in different organisms.
Homework Questions from Dr. LeProust:
What’s the most commonly used method for oligo synthesis currently?
Solid-phase oligonucleotide synthesis using phosphoramidite chemistry.
Why is it difficult to make oligos longer than 200nt via direct synthesis?
Due to the accumulation of small errors.
Why can’t you make a 2000bp gene via direct oligo synthesis?
It is difficult to make oligos longer than 200nt via direct synthesis due to error accumulation.
Homework Question from George Church:
[Using Google & Prof. Church’s slide #4] What are the 10 essential amino acids in all animals
and how does this affect your view of the “Lysine Contingency”?
The 10 essential amino acids are Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan, and Valine. This is interesting for the “Lysine Contingency” since animals cannot synthesize any of these essential amino acids (in high enough quantities), so they have to get them through their diet. Making dinosaurs reliant on external Lysine is just making them like any normal animal.
Part 2: Gel Art - Restriction Digests and Gel Electrophoresis
Our design
Our gel results
Part 3: DNA Design Challenge
3.1. Choose your protein.
I chose Antirepressor protein ant from Salmonella phage P22 since my lab is interested in antirepressors in bacteriophage and how they induce phage.
>sp|P03037|RANT_BPP22 Antirepressor protein ant OS=Salmonella phage P22 OX=10754 GN=ant PE=1 SV=1 MNSIAILEAVNTSYVPFNGQHVLTAMVAGVAYVAMKPVVDNIGLSWSSQVQKLLKMKDKFNYVDIDMVAGDMKKRLMGCIPLKKLNGWLFSINPEKVRADIRDKLIKYQEECFTVLYDYWTKGKAENPRKKTSVDERTPLRDAVNMLVSKKHLMYPEAYAMIHQRFNVESIEELEASQIPLAVEYIHRVVLEGEFIGKQEKKTNDLSAKEANSLVWLWDYANRSQALFRELYPAMRQIQSNYSGKCYDYGHEFSYIIGIARDVLINHTRDVDINEPDGPTNLSAWMRLKDKELPPSLHRY
3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.
Ant protein DNA sequence with Codon-Optimization ATGATGAATTCTATTGCTATTTTAGAAGCTGTTAATACTTCTTATGTTCCTTTTAATGGTCAACATGTTTTAACTGCTATGGTTGCTGGTGTTGCTTATGTTGCTATGAAACCTGTTGTTGATAATATTGGTTTATCTTGGTCTTCTCAAGTTCAAAAATTATTAAAAATGAAAGATAAATTTAATTATGTTGATATTGATATGGTTGCTGGTGATATGAAAAAACGTTTAATGGGTTGTATTCCTTTAAAAAAATTAAATGGTTGGTTATTTTCTATTAATCCTGAAAAAGTTCGTGCTGATATTCGTGATAAATTAATTAAATATCAAGAAGAATGTTTTACTGTTTTATATGATTATTGGACTAAAGGTAAAGCTGAAAATCCTCGTAAAAAAACTTCTGTTGATGAACGTACTCCTTTACGTGATGCTGTTAATATGTTAGTTTCTAAAAAACATTTAATGTATCCTGAAGCTTATGCTATGATTCATCAACGTTTTAATGTTGAATCTATTGAAGAATTAGAAGCTTCTCAAATTCCTTTAGCTGTTGAATATATTCATCGTGTTGTTTTAGAAGGTGAATTTATTGGTAAACAAGAAAAAAAAACTAATGATTTATCTGCTAAAGAAGCTAATTCTTTAGTTTGGTTATGGGATTATGCTAATCGTTCTCAAGCTTTATTTCGTGAATTATATCCTGCTATGCGTCAAATTCAATCTAATTATTCTGGTAAATGTTATGATTATGGTCATGAATTTTCTTATATTATTGGTATTGCTCGTGATGTTTTAATTAATCATACTCGTGATGTTGATATTAATGAACCTGATGGTCCTACTAATTTATCTGCTTGGATGCGTTTAAAAGATAAAGAATTACCTCCTTCTTTACATCGTTATTAA
3.3. Codon optimization.
Different organisms have different bias to certain tRNA which makes them quicker as translating certain codons. Therefore to make the most proteins the quickest, you need to optimize for the organism that will be producing the protein (is being engineered to make it). I have optimized for Salmonella typhimurium (LT2) since it is the only option I found in benchling from the Salmonella genus (which the phage infects).
Ant protein DNA sequence with Codon-Optimization ATGATGAACAGCATCGCGATTTTGGAGGCCGTGAATACGTCGTATGTCCCGTTTAATGGCCAGCATGTCCTGACCGCAATGGTAGCGGGCGTCGCATATGTGGCGATGAAGCCGGTTGTTGATAATATCGGTCTTAGTTGGTCGTCCCAGGTCCAAAAACTTCTGAAAATGAAAGATAAGTTCAACTATGTTGATATCGACATGGTTGCGGGCGATATGAAAAAAAGACTGATGGGCTGCATTCCGCTGAAGAAATTGAACGGGTGGCTCTTCTCCATAAATCCCGAAAAAGTACGAGCGGATATTCGTGACAAGCTGATCAAATATCAGGAAGAGTGCTTTACAGTACTTTACGACTATTGGACGAAAGGAAAAGCCGAGAACCCGCGTAAAAAAACGTCTGTGGACGAACGGACCCCGTTACGCGATGCGGTTAACATGCTCGTGAGCAAAAAACACCTGATGTACCCGGAAGCTTATGCTATGATCCATCAGCGCTTTAACGTGGAATCAATCGAGGAACTGGAAGCCTCGCAAATTCCATTAGCCGTCGAATACATTCACCGCGTGGTGCTCGAAGGTGAGTTTATTGGCAAACAGGAAAAGAAGACCAATGATTTGTCCGCAAAAGAGGCCAACAGCCTGGTGTGGCTATGGGACTACGCCAATCGCAGCCAGGCTCTGTTTCGTGAACTGTACCCGGCGATGCGTCAGATTCAAAGCAATTATAGCGGAAAATGTTATGATTATGGCCATGAATTCTCTTATATCATTGGGATCGCGCGTGACGTATTAATTAATCATACCCGCGATGTCGATATAAACGAACCTGACGGTCCAACTAACCTGAGTGCGTGGATGCGGCTGAAAGATAAAGAGCTGCCTCCCTCACTGCACCGCTACTAA
3.4. You have a sequence! Now what?
The DNA can be transcribed and translated into the protein by inserting it into a plasmid which is put into the host (bacteria) through methods like chemical transformation or electroporation (which interupt the stability of the cell membrane), and then the host starts expressing it like it would for one of its own proteins.
Part 4: Prepare a Twist DNA Synthesis Order
Benchling annotation
Plasmid
Part 5: DNA Read/Write/Edit
5.1 DNA Read
(i) What DNA would you want to sequence (e.g., read) and why?
I am interested in sequencing bacteriophage and environmental samples. Bacteriophage are becoming a possibility for drug resistant bacteria treatments, and they can be a vector in bio engineering. Their prevalence in the environment and interactions with bacteria also make it interesting to study metagenomic samples with and without phage.
(ii) In lecture, a variety of sequencing technologies were mentioned. What technology or technologies would you use to perform sequencing on your DNA and why?\
In the past, we have used illumina (NGS) and plasmidsaurus (nanopore) for phage. Plasmidsaurus is quicker and less expensive but has had variable results. Metagenomics also uses NGS and sometimes nanopore (can be more portable for lab work).
NGS is second-gen, nanopore is third-gen.
Input is the library which is fragmented extracted DNA which has gone through end repair, a-tailing, and adapter ligation, also possibly pcr if the target sequence needs to be amplified.
Illumina works by isothermically amplifying the fragments, fragment strands binding to oligos on one end, polymerase creating the reverse strands of the fragments, and then bridge amplification repeating over and over for all the fragments. The reverse strands are discarded and then the forward strands are sequenced through fluorescently tagged nucleotides being added one by one, with the signals being read for each added base and associated with the length.
The output of illumina is millions or more of short reads. The output for plasmidsaurus is long reads or whole plasmid.
5.2 DNA Write
(i) What DNA would you want to synthesize (e.g., write) and why?
I think that DNA encoded sensors are interesting. With a background in computing, I can see a lot of places where similar principals would apply and biological sensors could be useful (e.g. water testing, health testing, soil testing, working with hardware to give timelines, etc.).
(ii) What technology or technologies would you use to perform this DNA synthesis and why?\
Enzymatic DNA Synthesis would be good to use since it is more sustainable (and better for longer single strands), but it would be likely easier to start with phosphoramidite method synthesis which is industry standard and more cost-effective (for now).
The essential steps of the phosphoramidite method for oligonucleotide synthesis are
Deprotection (acid removes protecting group to expose hydroxyl group)
Coupling (nucleoside phosphoramidite is activated and added to hydroxyl group)
Capping (unreacted hydroxyl groups are “capped” to prevent further nucleotide addition)
Oxidation/Sulfurization (phosphite triester linkage is converted into stable pentavalent phosphotriester bond)
Limitations of the phosphoramidite method include length (only 100-200bp possible) and having way less sustainability (toxic organic waste is generated).
5.3 DNA Edit
(i) What DNA would you want to edit and why?
I think more can be done for flora, especially in the accessibility of parts (such as seeds) with edits. I think that in human medicine, preventatives and sensors are interesting. My aunt was diagnosed with stage 4 liver cancer in December with no prior warning. It would be great to have better ways to detect and understand silent conditions.
(ii) What technology or technologies would you use to perform these DNA edits and why?\
CRISPR Cas12a can be useful for biosensors, and bacterium mediated transfer and engineered phage (through plasmid transformation) are of interest for targeting plants.
CRISPR Cas12a processes its own precursor (unlike cas9) into crRNA which combines with the enzyme to form ribonucleoprotein (RNP) complexes. The complex recognizes and binds to a specific Protospacer Adjacent Motif (PAM) site, then breaks both strands (forms sticky ends) and starts breaking down ssDNA close by.
Preperation includes selecting the target sequence downstream of a PAM and designing the crRNA.
Limitations include requiring a specific PAM and lower efficiency than cas9 in some mammalian cells
Using custom design tools (trying out stippling, different dot sizes)
Python Files
What the different dot design looks like after programming so far (still need to scale a bit)
AI Usage documentation
I utilized Claude Opus 4.6 to help program a web app to assign values (light-dark as 0-8) to pixels of an image so that it could be represented in different dot sizes or clustering on a petri dish. I also used the model to help scale my programming and cordinates to the proper plate size. Claude models work well for general programming related tasks, but sometimes struggle with nailing down details (e.g. it worked out the logic for pixel calculations very well, but struggled to fix the display attributes and relative sizing)
The paper by Dufour et al. presents and evaluates using liquid handling robots for high-throughput phage susceptibility testing. This is important since as bacteriophage are used more for phage therapy and other applications, single phage and cocktail testing is used to determine the best phage for the particular target, but can be very time consuming to test manually. The method with the liquid handling robot was found to have lower variance and very similar mean results to the manual assays.
Final Project Lab Automation
For my final independent project I would like to use the Opentron T2 to run a large range of phage susceptibility tests, similar to my choosen paper. For my more computational project ideas this could be used to validate phage-host range results or possibly be done in the future with the edited phage designs, and for my phage sensor idea this would be done with the transformed bacteria to gather the luminance-titer results.
