Week 1 HW: Principles and Practices

1. First, describe a biological engineering application or tool you want to develop and why. The engineering of extracted hematopoietic stem cells so their B-cell progeny produces bnAbs (broadly neutralizing antibodies), so after exposure to an immunogen (a highly mutable virus like HIV, Influenza, and so on) they can provide protection for a long time after autologous engraftment.

The reason why I want to develop this is because it is near within our reach to be able to create a method of viral protection against a lot of viruses which we thought we’d never be able to get rid of.

2. 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.

Governance/policy goal: Trials for healthiness.

First and foremost: the evaluation if this should be something to be researched, weighing the benefits and the possible risks of bringing such tools into the world, the same way that chiral lifeforms was evaluated.

Secondly: biological safety for the patient. Rigorous and extensive research, as it goes for any kind of treatment! Specifically, see if this treatment leads to the development of an autoimmunity, or causes any harm in trials using an in-vivo model, then murine model, then human model.

3. Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”).

–First governance action: Authorization by prescription/indication

Purpose: To ensure this therapy doesn't drift abroad, it is required to make sure that only people go through the same approval process as gene therapies or CAR-T cell therapy.
Design: To make it work, we'd need FDA approval for risk groups in which conventional vaccines (if any were to be approved) or drugs like lenacapavir do not work. Once that's done, this immunotherapy could be standarized in capable hospitals. Hopefully can be funded by government, alternatively, donations, or both.
Assumptions: Assuming that non-authorized use is detectable/enforceable.
Risks of Failure & “Success”: Failure would be slowing down their the translational part of immunotherapy, success would be the exact opposite, but could lead to 'medical tourism'.

–Second governance action: Long term surveillance

Purpose: To ensure no harm happens off-target and there are no adverse effects, there is the need for long term surveillance.
Design: Hospitals collect periodic data, which incude checkups for any symptoms of autoimmunity. Funded by the patient's health insurance.
Assumptions: Data is secured and used ethically. 
Risks of Failure & “Success”: Underfunded registries may lead to loss of the follow ups, which would lead to (if present) any effect going undetected. Success would lead to privacy concerns.

–Third governance action: Contain by design

Purpose: Standarize the protocol (as much as it can be, considering this is a personalized immunotherapy) to ensure it doesn't go off-target, 
Design: by establishing the gene(s) to be inserted, promoter(s), and the locus/loci to target. Lastly, a kill switch in case the immunotherapy goes wrong, such as an over-expressed protein to target with antibodies.
Assumptions: Assumes that the protocol and go wrong can also be 'reversed' by the addition of a kill switch.
Risks of Failure & “Success”: By design there's the assumption that the safeguards will generate confidence (that could be false confidence if the design does not work). Success can bring costs higher by adding complexity (particularly the kill switch). 

4. Next, score (from 1-3 with, 1 as the best, or n/a) each of your governance actions against your rubric of policy goals. The following is one framework but feel free to make your own:

5. 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. For this, you can choose one or more relevant audiences for your recommendation, which could range from the very local (e.g. to MIT leadership or Cambridge Mayoral Office) to the national (e.g. to President Biden or the head of a Federal Agency) to the international (e.g. to the United Nations Office of the Secretary-General, or the leadership of a multinational firm or industry consortia). These could also be one of the “actor” groups in your matrix.

Definitely the third option, contain by design. Not only because of the scoring, but because I believe the first 2 options already are a must, given how common the those logistics are in medical proceedures alike. I believe the trade-offs mostly come from the risks (of failure and the ones that come from success in every option) rather than from the operation itself. I believe this would be the most relevant to academic medical centers.

–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. This should be included on your class page for this week.

The possibility of the immunotherapy causing autoimmunity, after a long time. I wasn’t accounting on the duration of the immune cells in the body. The governance actions appropriate for those issues would be option 3 and/or 2.

Assignment (Week 2 Lecture Prep) — DUE BY START OF FEB 10 LECTURE

Homework Questions from Professor Jacobson

1. 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?

The error rate is 1:10 to the power of 6! This doesn’t compare much to the length of the human genome, which is 3:10 to the power of 9. Biology deals with this through proofreading activity; through 3’ to 5’ exonuclease activity.

2. 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?

So the average human protein has 1036 base pairs, and the average aminoacid has 4 ways of synthesis, and because this is permutations (how many combinations there are), it would be 4 to the power of 1036. There can be many reasons such as, unoptimal within cellular context (such as, pH is not optimal for the final product when using a certain combination of aminoacids, and so the protein’s faulty, but it would be for a different combination of aminoacids in which the pH is actually suitable for the protein, so the second one would be conserved), another one reason could be rare tRNA’s that would slow down the synthesis, paradoxically, it could also be a combination of only optimal codons, which would make ribosome speed go beyond its capability, as in, make it go too fast to the point where the folding does not occur correctly (this last one I read in a paper that I’d like to be able to find again, it was something about mechanistic properties of synthesis from the ribosome).

Homework Questions from Dr. LeProust

1. What’s the most commonly used method for oligo synthesis currently? Phosphodiester method, I believe that’s what the majority has access to.

2. Why is it difficult to make oligos longer than 200nt via direct synthesis? Because each nucleotide addition requires all steps such as coupling, capping, oxidation, deblocking, at around 200 cycles is where it begins to truncate

3. Why can’t you make a 2000bp gene via direct oligo synthesis? Because truncation seems to happen after 200 cycles. There are methods that can go up to 500nt, but that’s pushing it!

Homework Question from George Church

1. Choose ONE of the following three questions to answer; and please cite AI prompts or paper citations used, if any.

[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”? No AI. (Kansas State University, n. d). The 10 aminoacids are lysine, methionine, tryptophan, threonine, valine, isoleucine, leucine, arginine, histidine and phenylalanine. Peculiar, that does nothing… and even if they were to do that, couldn’t the dinosaurs just get their lysine from regular animals or plants? I think this contingency plan would work if it was instead a deletion or anything that were to stop the translation of the enzyme(s) that synthesize an aminoacid or metabolite that is produced by dinosaurs (and is rarely ever found in diet).

Bibliography:

Kansas State University. (n. d.). Essential and non-essential amino acids. Animal Sciences And Industry. https://www.asi.k-state.edu/extension/swine/swinenutritionguide/general_nutrition_principles/essentialandnonessentialaminoacids.html

Week 2 HW: DNA Read, Write, & Edit

Part 0: Basics of Gel Electrophoresis

Attend or watch all lecture and recitation videos. Optionally watch bootcamp.

Done :checkmark:

Part 1: Benchling & In-silico Gel Art

See the Gel Art: Restriction Digests and Gel Electrophoresis protocol for details. Overview:

Make a free account at benchling.com
Import the Lambda DNA.
Simulate Restriction Enzyme Digestion with the following Enzymes:
    EcoRI
    HindIII
    BamHI
    KpnI
    EcoRV
    SacI
    SalI
Create a pattern/image in the style of Paul Vanouse’s Latent Figure Protocol artworks.
You might find Ronan’s website a helpful tool for quickly iterating on designs.

I title it “A greeting..”

Part 2: Benchling & In-silico Gel Art

Unable to do, lack the lab access.

Part 3: DNA Design Challenge

3.1. Choose your protein.

Which protein have you chosen and why?

I have chosen Lactose. I wanted to do Lactase instead, but it seemed to big.

sp|P00709|LALBA_HUMAN Alpha-lactalbumin OS=Homo sapiens OX=9606 GN=LALBA PE=1 SV=1 MRFFVPLFLVGILFPAILAKQFTKCELSQLLKDIDGYGGIALPELICTMFHTSGYDTQAI VENNESTEYGLFQISNKLWCKSSQVPQSRNICDISCDKFLDDDITDDIMCAKKILDIKGI DYWLAHKALCTEKLEQWLCEKL

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

Alpha-lactalbumin DNA sequence

atgcgcttttttgtgccgctgtttctggtgggcattctgtttccggcgattctggcgaaa cagtttaccaaatgcgaactgagccagctgctgaaagatattgatggctatggcggcatt gcgctgccggaactgatttgcaccatgtttcataccagcggctatgatacccaggcgatt gtggaaaacaacgaaagcaccgaatatggcctgtttcagattagcaacaaactgtggtgc aaaagcagccaggtgccgcagagccgcaacatttgcgatattagctgcgataaatttctg gatgatgatattaccgatgatattatgtgcgcgaaaaaaattctggatattaaaggcatt gattattggctggcgcataaagcgctgtgcaccgaaaaactggaacagtggctgtgcgaa aaactg

3.3. Codon optimization.

Alpha-lactalbumin DNA sequence Optimized

ATGCGATTCTTCGTCCCCCTGTTTCTGGTCGGTATTCTGTTCCCCGCCATCCTCGCCAAGCAGTTCACCAAGTGCGAGCTGTCCCAGCTGCTGAAGGACATCGACGGATACGGTGGTATCGCCCTGCCCGAGCTGATTTGCACCATGTTCCACACCTCTGGATACGACACCCAGGCCATCGTCGAGAACAACGAGTCCACTGAGTACGGCCTGTTCCAGATTTCCAACAAGCTGTGGTGCAAGTCTTCTCAGGTTCCTCAGTCCCGAAACATCTGCGACATTTCTTGCGACAAGTTCCTGGACGACGACATCACCGACGACATCATGTGCGCCAAGAAGATTCTGGACATCAAGGGTATCGACTACTGGCTGGCCCACAAGGCCCTGTGCACCGAGAAGCTGGAGCAGTGGCTGTGCGAGAAGCTG

In your own words, describe why you need to optimize codon usage. Which organism have you chosen to optimize the codon sequence for and why?

Codon optimization is needed in case a sequence contains too many rare tRNAs (which, may be common from the original organism), which could mess up with protein translation; it also may impact the protein’s stability depending on the cellular context, specifically, the pH, so having compatible codon’s for once the protein’s done is also something to keep in mind.

3.4. You have a sequence! Now what?

What technologies could be used to produce this protein from your DNA? Describe in your words the DNA sequence can be transcribed and translated into your protein. You may describe either cell-dependent or cell-free methods, or both.

First, it needs a promoter, and a terminator; once that’s edited into the sequence, it can be inserted into a yeast (I chose yeast and not a bacteria because there are some post-translational-modifications that bacteria is unable to do!)

Oh, also, it would be a plasmid. In order to insert this plasmid, a method that can be used is Heat Shock of Yarrowia lipolytica (forgive me if I forget to name the organism in italics). Would just need to follow the protocol, but also design a selection method, such as the introduction of an antibiotic-resistant gene, or the usage of a strain that lacks the capability of producing a metabolite that is essential for metabolism, transform, and then pass it onto a medium that also does not have said metabolite.

Part 4: Prepare a Twist DNA Synthesis Order

4.1. Create a Twist account and a Benchling account

Done!

4.2. Build Your DNA Insert Sequence

https://benchling.com/s/seq-JUNnre8HZRwtYLbAV7ER?m=slm-NLI7GfaPtF5eARNkdXdJ

Done also!

4.3. On Twist, Select The “Genes” Option

Mhm

4.4. Select “Clonal Genes” option

Yep

4.5. Import your sequence

Done!

4.6. Choose Your Vector

I chose pTwist Amp High Copy - (2221bp)

My, hopefully functional, plasmid..

Part 5: DNA Read/Write/Edit

5.1 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, biobank).

I think something interesting would be the DNA of all, if not, multiple flowers that produce the color blue. Blue is quite a rare color in nature, and it would be wonderful if it could be figured out what genes are responsible for the blue of the flower… the reason why is because, with genetic engineering, surely blue cotton could be possible!

(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? Also answer the following questions:

Illumina’s sequencing-by-synthesis. It has a great output and, because plant genomes are immense, I think a technology that can do a whole-genome sequencing would be the best fit for the DNA I want to sequence as answered above.

Is your method first-, second- or third-generation or other? How so?

Second generation! Because it’s a high output and massive-in-parallel sequencing method

What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.

Purified plant DNA, quantify DNA (with nanodrop), fragmentation, NGS protocol (usage of adapters and then the fragments are amplified with PCR. Then this library is ready for the sequencer).

What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?

Using the sequencer, the DNA fragments are absorbed by the flow cell, new strands of DNA (one base at a time) are synthesized with fluorescent labeled nucleotides. Given that each nucleotide will have a different color, each new base added will be captured.

What is the output of your chosen sequencing technology?

A digital file with reads, which are these sequences that are meant to be put together to reconstruct the whole genome.

5.2 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.), to art (DNA origamis). 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 want to synthesize a mRNA vaccine that uses the principle of those superadjuvant HIV vaccines, but the immunogen would be a cancer molecule. It would be interesting to see the effects of bnAbs against cancer.

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

A DNA plasmid into RNA with the use of phage RNA polymerase. The reason why is because it is an mRNA vaccine, so that polymerase is very essential.

Also answer the following questions:

What are the essential steps of your chosen sequencing methods?

Design the plasmid template with compatible elements like the promoter for the phage RNA polymerase. Then, do in vitro transcription.

What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?

This is a method that is relatively simple and does not really require much speed given that it is a small synthesis (in terms of nucleotides), accurate-wise it can be ~70-80% because capping sometimes can be incomplete. Since it is an enzymatic in vitro process, it is suitable to be scaled up pretty quickly.

5.3 DNA Edit

(i) 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 for human health, development, and even human longevity and human augmentation. DNA editing is also already commonly leveraged for flora and fauna, for example in nature conservation efforts, (animal/plant restoration, de-extinction), or in agriculture (e.g. plant breeding, nitrogen fixation). What kinds of edits might you want to make to DNA (e.g., human genomes and beyond) and why?

I would want to edit the p53 gene. I believe that elephants do not have risky rates of cancer despite their size/number of cells, and that’s because (If I am not misremembering) they have a bunch of p53 copies. Reason I would do this is for the better health of a lot of people.

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

CRISPR. Because I think this would be the safest option given how many gene editing therapies that use CRISPR are being approved lately.

Also answer the following questions:

How does your technology of choice edit DNA? What are the essential steps?

CRISPR-Cas9 uses a guide RNA that matches a specific DNA sequence, and it has the enzyme Cas9 which is directed by said guide to the target. This enzyme makes a double-strand break, and then the cell tries to repair this break, while it is repairing it, there can be the insertion of a corrected sequence if a repair template is provided.

What preparation do you need to do (e.g. design steps) and what is the input (e.g. DNA template, enzymes, plasmids, primers, guides, cells) for the editing?

Target DNA is identified in order to design the guide RNA. The input includes Cas9, guide RNA and donor DNA template.

What are the limitations of your editing methods (if any) in terms of efficiency or precision?

I’m not aware of any as CRISPR seems to be described as a very efficient and precise gene editing tool in literature.

Week 3 HW: Lab Automation

Homework

Assignment: Python Script for Opentrons Artwork — DUE BY YOUR LAB TIME!

Committed Listeners Required

Your task this week is to Create a Python file to run on an Opentrons liquid handling robot.

Review this week’s recitation and this week’s lab for details on the Opentrons and programming it.
Generate an artistic design using the GUI at opentrons-art.rcdonovan.com.
Using the coordinates from the GUI, follow the instructions in the HTGAA26 Opentrons Colab to write your own Python script which draws your design using the Opentrons.
    You may use AI assistance for this coding — Google Gemini is integrated into Colab (see the stylized star bottom center); it will do a good job writing functional Python, while you probably need to take charge of the art concept.
    If you’re a proficient programmer and you’d rather code something mathematical or algorithmic instead of using your GUI coordinates, you may do that instead.
Ask for help early!

If you are having any trouble with scripting, contact your TAs as soon as possible for help.
Do not wait until your scheduled robot time slot or you may not be able to complete this assignment!
If the Python component is proving too problematic even with AI and human assistance, download the full Python script from the GUI website and submit that:
Use the download icon pointed to by the red arrow in this diagram.

Use the download icon pointed to by the red arrow in this diagram.
If you use AI to help complete this homework or lab, document how you used AI and which models made contributions.
Sign up for a robot time slot if you are at MIT/Harvard/Wellesley or at a Node offering Opentrons automation. The Python script you created will be run on the robot to produce your work of art!
    At MIT/Harvard? Lab times are on Thursday Feb.19 between 10AM and 6PM.
    At other Nodes? Please coordinate with your Node.

Submit your Python file via this form.

https://opentrons-art.rcdonovan.com/?id=86flkdas3nws5bs

Post-Lab Questions — DUE BY START OF FEB 24 LECTURE

One of the great parts about having an automated robot is being able to precisely mix, deposit, and run reactions without much intervention, and design and deploy experiments remotely.

For this week, we’d like for you to do the following:

Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.

Bryant Jr, J. A., Kellinger, M., Longmire, C., Miller, R., & Wright, R. C. (2023). AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots. Synthetic Biology, 8(1), ysac032.

They published an open-source Python software package called “AssemblyTron” and it automates DNA assembly worfklows using the Opentrons OT-2 liquid handling opentrons.. it can automate PCR setupts, gradient optimization, Golden Gate assembly and homology based in vivo assembly.

Write a description about what you intend to do with automation tools for your final project. You may include example pseudocode, Python scripts, 3D printed holders, a plan for how to use Ginkgo Nebula, and more. You may reference this week’s recitation slide deck for lab automation details.

While your description/project idea doesn’t need to be set in stone, we would like to see core details of what you would automate. This is due at the start of lecture and does not need to be tested on the Opentrons yet.

Example 1: You are creating a custom fabric, and want to deposit art onto specific parts that need to be intertwined in odd ways. You can design a 3D printed holder to attach this fabric to it, and be able to deposit bio art on top. Check out the Opentrons 3D Printing Directory.

Example 2: You are using the cloud laboratory to screen an array of biosensor constructs that you design, synthesize, and express using cell-free protein synthesis.

Echo transfer biosensor constructs and any required cofactors into specified wells.
Bravo stamp in CPFS reagent master mix into all wells of a 96-well / 384-well plate.
Multiflo dispense the CFPS lysate to all wells to start protein expression.
PlateLoc seal the plate.
Inheco incubate the plate at 37°C while the biosensor proteins are synthesized.
XPeel remove the seal.
PHERAstar measure fluorescence to compare biosensor responses.

Idea 1, The engineering of extracted hematopoietic stem cells so their B-cell progeny produces bnAbs and after exposure to an HIV immunogen they can provide protection for a long time after autologous engraftment: AUTOMATION

So, with python I could generate safety kill-switch modules. I could Echo transfer plasmids into 96-well plates, Inhecho incubate the cells, automate ELISA plate reader for measuring antibody secretion.

Idea 2, Geroprotective psilocybin and hallucinogenic blockade/evasion: A liposome that evades the BBB with a geroprotective carrier: AUTOMATION

Automated drug loading quantification via plate reader,

Idea 3, Bioluminiscent trees: AUTOMATION

Echo transfer DNA fragments into 96-well plates, automate Agrobacterium transformation mixes, and potentially a Python image analysis that quantifies glow intensity.

There’s a lot that can be automated; it is not my strength just yet.

Final Project Ideas — DUE BY START OF FEB 24 LECTURE

Committed Listeners Required

As explained in this week’s recitation, add 1-3 slides in your Node’s section of this slide deck with 3 ideas you have for an Individual Final Project. Be sure to put your name, city, and country on your slide!