Week 1 — Principles & Practices

This week lays the foundation for ethics, safety, and governance in biotechnology — and we get hands-on with lab basics.

Homework — DUE BY START OF FEB 10 LECTURE

Documentation

Make sure to document every step of the in-silico and lab experiments. Make sketches, screenshots, notes, drawings - anything that helps you - and others understand the experiment.

Your Documentation should help you - and others - to understand the topic. Don’t be afraid to add things that don’t work. Show your failures - and how you overcame them. Your Documentation should be a description of the amazing journey you are on!

Overview

Ethics, safety, and security are essential considerations throughout (and beyond!) this class. We have therefore designed the Class Assignment this week to give you a strong foundation, and then will ask you to reflect each week and in the design of your final project.

Class Assignment — DUE BY START OF FEB 10 LECTURE

1. First, describe a biological engineering application or tool you want to develop and why. This could be inspired by an idea for your HTGAA class project and/or something for which you are already doing in your research, or something you are just curious about.
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. Below is one example framework (developed in the context of synthetic genomics) you can choose to use or adapt, or you can develop your own. The example was developed to consider policy goals of ensuring safety and security, alongside other goals, like promoting constructive uses, but you could propose other goals for example, those relating to equity or autonomy.
3. Next, describe at least three different potential governance “actions” by considering the four aspects below (Purpose, Design, Assumptions, Risks of Failure & “Success”). Try to outline a mix of actions (e.g. a new requirement/rule, incentive, or technical strategy) pursued by different “actors” (e.g. academic researchers, companies, federal regulators, law enforcement, etc). Draw upon your existing knowledge and a little additional digging, and feel free to use analogies to other domains (e.g. 3D printing, drones, financial systems, etc.).
   1. Purpose: What is done now and what changes are you proposing?
   2. Design: What is needed to make it “work”? (including the actor(s) involved - who must opt-in, fund, approve, or implement, etc)
   3. Assumptions: What could you have wrong (incorrect assumptions, uncertainties)?
   4. Risks of Failure & “Success”: How might this fail, including any unintended consequences of the “success” of your proposed actions?
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.

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.

Assignment (Final Project) – Due as part of your Final Project presentation (not Feb 10)

As part of your final project, design one or more strategies to ensure that your project, and what it enables, contributes to growing an ethical biological future.

Assignment (Lab Preparation) — DUE BY START OF FEB 10 LECTURE

Lab Training (failure to complete this will jeopardize your acceptance into the course)

• Complete Lab Specific Training in Person.
• Complete Safety Training in Atlas
  • Navigate to atlas.mit.edu and on the right-hand side, click “Learning Center”
  • Head to the Course Catalog and find the following two courses:
    • General Biosafety for Researchers (EHS00260w)
    • Managing Hazardous Waste (EHS00501w)

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

In preparation for Week 2’s lecture on “DNA Read, Write, and Edit," please review these materials:

1. Lecture 2 slides as posted below.
2. The associated papers that are referenced in those slides.

In addition, answer these questions in each faculty member’s section:

Homework Questions from Professor Jacobson: [Lecture 2 slides]

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?
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?

Homework Questions from Dr. LeProust: [Lecture 2 slides]

1. What’s the most commonly used method for oligo synthesis currently?
2. Why is it difficult to make oligos longer than 200nt via direct synthesis?
3. Why can’t you make a 2000bp gene via direct oligo synthesis?

Homework Question from George Church: [Lecture 2 slides]

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

1. [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”?
2. [Given slides #2 & 4 (AA:NA and NA:NA codes)]   What code would you suggest for AA:AA interactions?
3. [(Advanced students)]   Given the one paragraph abstracts for these real 2026 grant programs sketch a response to one of them or devise one of your own:
   • https://arpa-h.gov/explore-funding/programs/boss
   • https://www.darpa.mil/research/programs/smart-rbc
   • https://www.darpa.mil/research/programs/go

Assignment (Your HTGAA Website) — DUE BY START OF FEB 10 LECTURE

1. Begin personalizing your HTGAA website in in https://edit.htgaa.org/, starting with your homepage — fill in the template with information about yourself, or remove what’s there and make it your own. Be creative!
2. As with all assignments in HTGAA, be sure to write up every part of this Homework on your HTGAA website in order to receive credit.

Week 2 — DNA Read, Write, & Edit

This week explores the read–write–edit toolkit: sequencing and synthesis workflows, restriction digests and gel electrophoresis, and early genome-editing frameworks.

Homework — DUE BY FEB 17 2PM MIT TIME

Documentation

Make sure to document every step of the in-silico and lab experiments. Make sketches, screenshots, notes, drawings… anything that helps you - and others - understand the experiment.

Your documentation should help you - and others - to understand the topic. Don’t be afraid to add things that don’t work. Show your failures - and how you overcame them. Your Documentation should be a description of the amazing journey you are on!

Part 0: Basics of Gel Electrophoresis

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

Part 1: Benchling & In-silico Gel Art

See this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis” 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!

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

In the wet-lab perform the lab experiment you designed in Part 1 and outlined in this week’s lab protocol “Gel Art: Restriction Digests and Gel Electrophoresis”.

Part 3: DNA Design Challenge

3.1. Choose your protein.

In recitation, we discussed that you will pick a protein for your homework that you find interesting. Which protein have you chosen and why? Using one of the tools described in recitation (NCBI, UniProt, google), obtain the protein sequence for the protein you chose.

[Example from our group homework, you may notice the particular format — The example below came from UniProt]

>sp|P03609|LYS_BPMS2 Lysis protein OS=Escherichia phage MS2 OX=12022 PE=2 SV=1 METRFPQQSQQTPASTNRRRPFKHEDYPCRRQQRSSTLYVLIFLAIFLSKFTNQLLLSLL EAVIRTVTTLQQLLT

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

The Central Dogma discussed in class and recitation describes the process in which DNA sequence becomes transcribed and translated into protein. The Central Dogma gives us the framework to work backwards from a given protein sequence and infer the DNA sequence that the protein is derived from. Using one of the tools discussed in class, NCBI or online tools (google “reverse translation tools”), determine the nucleotide sequence that corresponds to the protein sequence you chose above.

[Example: Get to the original sequence of phage MS2 L-protein from its genome phage MS2 genome - Nucleotide - NCBI]

Lysis protein DNA sequence
atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa

3.3. Codon optimization.

Once a nucleotide sequence of your protein is determined, you need to codon optimize your sequence. You may, once again, utilize google for a “codon optimization tool”. 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?

[Example from Codon Optimization Tool | Twist Bioscience while avoiding Type IIs enzyme recognition sites BsaI, BsmBI, and BbsI]

Lysis protein DNA sequence with Codon-Optimization
ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA

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.

3.5. [Optional] How does it work in nature/biological systems?

1. Describe how a single gene codes for multiple proteins at the transcriptional level.
2. Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!! See example below.

[Example shows the biomolecular flow in central dogma from DNA to RNA to Protein] Special note that all “T” were transcribed into “U” and that the 3-nt codon represents 1-AA.

Part 4: Prepare a Twist DNA Synthesis Order

This is a practice exercise, not necessarily your real Twist order!

4.1. Create a Twist account and a Benchling account

4.2. Build Your DNA Insert Sequence

For example, let’s make a sequence that will make E. coli glow fluorescent green under UV light by constitutively (always) expressing sfGFP (a green fluorescent protein):

In Benchling, select New DNA/RNA sequence

Give your insert sequence a name and select DNA with a Linear topology (this is a linear sequence that will be inserted into a circular backbone vector of our choosing).

Go through each piece of the given DNA sequences highlighted below (Promoter, RBS, Start Codon, Coding Sequence, His Tag, Stop Codon, Terminator) and paste the sequences into the Benchling file one after the other (replacing the coding sequence with your codon optimized DNA sequence of interest!). Each time you add a new piece of the sequence, make sure to annotate by right clicking over the sequence and creating an annotation that describes what each piece (e.g., Promoter, RBS, etc.) is (see image below).

• Promoter (e.g. BBa_J23106): TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC
• RBS (e.g. BBa_B0034 with spacers for optimal expression): CATTAAAGAGGAGAAAGGTACC
• Start Codon: ATG
• Coding Sequence (your codon optimized DNA for a protein of interest, sfGFP for example): AGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA
• 7x His Tag (Let’s add a 7×His tag at the C-terminus of the protein to enable protein purification from E. coli): CATCACCATCACCATCATCAC
• Stop Codon: TAA
• Terminator (e.g. BBa_B0015): CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

Once you’ve completed this, click on Linear Map to preview the entire sequence. If you intend to have a TA review a sequence in the future, this is a good way to verify that all sections are annotated!

This is not required for this exercise, but to share your design with others, please ensure that link sharing is turned on! (Optional) Share your final sequence link with a TA for review!

This insert sequence you built is commonly referred to as an expression cassette in molecular biology (a sequence you can drop into any vector and it’ll perform its function). Go ahead and download the FASTA file for the sequence you made.

It’s helpful to visualize DNA designs using SBOL Canvas (Synthetic Biology Open Language) to convey your designs. Here’s an example of what you just annotated in Benchling:

4.3. On Twist, Select The “Genes” Option

4.4. Select “Clonal Genes” option

For this demonstration, we’ll choose Clonal Genes. You’ll select clonal genes or gene fragments depending on your final project.

Historically, HTGAA projects using clonal genes (circular DNA) have reached experimental results 1-2 weeks quicker because they can be transformed directly into E. coli without additional assembly.

Gene fragments (linear DNA) offer greater design flexibility but typically require an assembly or cloning step prior to transformation. An advantage is If designed with the appropriate exonuclease protection, gene fragments can be used directly in cell-free expression.

4.5. Import your sequence

You just took an amino acid sequence of interest and converted it into DNA, codon optimized it, and built an expression cassette around it! Choose the Nucleotide Sequence option and Upload Sequence File to upload your FASTA file.

4.6. Choose Your Vector

Since we’re ordering a clonal gene, you will need to refer to Twist’s Vector Catalog to choose your circular backbone. You can think of this as taking your linear expression cassette for your protein of interest, and completing the rest of the circle!

The backbone confers many special properties like antibiotic resistance, an origin of replication, and more. Discuss with your node to decide on appropriate antibiotic options. At MIT/Harvard, you can use Ampicillin, Chloramphenicol, or Kanamycin resistance.

Twist vectors do not contain restriction sites near the insert fragment, so make sure to flank your design with cut sites if you are intending to extract this DNA insert fragment later.

For this demonstration, choose a Twist cloning vectors like pTwist Amp High Copy.

Click into your sequence and select download construct (GenBank) to get the full plasmid sequence:

Go back to your Benchling account. Inside of a folder, click the import DNA/RNA sequence button and upload the GenBank file you just downloaded.

This is the plasmid you just built with your expression cassette included. Congratulations on building your first 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).

(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:

1. Is your method first-, second- or third-generation or other? How so?
2. What is your input? How do you prepare your input (e.g. fragmentation, adapter ligation, PCR)? List the essential steps.
3. What are the essential steps of your chosen sequencing technology, how does it decode the bases of your DNA sample (base calling)?
4. What is the output of your chosen sequencing technology?

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! :)

See some famous examples of DNA design

(ii) What technology or technologies would you use to perform this DNA synthesis and why?
Also answer the following questions:

1. What are the essential steps of your chosen sequencing methods?
2. What are the limitations of your sequencing method (if any) in terms of speed, accuracy, scalability?

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?

(ii) What technology or technologies would you use to perform these DNA edits and why?
Also answer the following questions:

1. How does your technology of choice edit DNA? What are the essential steps?
2. 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?
3. What are the limitations of your editing methods (if any) in terms of efficiency or precision?

Week 3 — Lab Automation

This week we get hands-on (or at least code-on) with pipetting robots.

Homework

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

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

0. Review this week’s recitation and this week’s lab for details on the Opentrons and programming it.
1. Generate an artistic design using the GUI at opentrons-art.rcdonovan.com.
2. 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!

3. 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:

   

4. If you use AI to help complete this homework or lab, document how you used AI and which models made contributions.
5. 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.
6. Submit your Python file via this form.

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:

1. Find and describe a published paper that utilizes the Opentrons or an automation tool to achieve novel biological applications.
2. 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.

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

Final Project Ideas — DUE BY START OF FEB 24 LECTURE

As explained in this week’s recitation, add 1-3 slides with 3 ideas you have for an Individual Final Project in the appropriate slide deck for MIT/Harvard/Wellesley students or for Commited Listeners. Be sure to put your name on your slide(s); for CLs, also put your city and country on your slide(s) and be sure you’re putting your slide(s) in your Node’s section of the deck.

Week 4 — Protein Design Part I

This week focuses on how sequence, structure, and energetics can be modeled and manipulated to create or optimize proteins with specified functions.

Homework: Protein Design I — DUE BY START OF MAR 3 LECTURE

Objective:

1. Learn basic concepts:
   • amino acid structure
   • 3D protein visualization
   • the variety of ML-based design tools
2. Brainstorm as a group how to apply these tools to engineer a better bacteriophage (setting the stage for the final project).

Part A. Conceptual Questions

Answer any NINE of the following questions from Shuguang Zhang: (i.e. you can select two to skip)

1. How many molecules of amino acids do you take with a piece of 500 grams of meat? (on average an amino acid is ~100 Daltons)
2. Why do humans eat beef but do not become a cow, eat fish but do not become fish?
3. Why are there only 20 natural amino acids?
4. Can you make other non-natural amino acids? Design some new amino acids.
5. Where did amino acids come from before enzymes that make them, and before life started?
6. If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?
7. Can you discover additional helices in proteins?
8. Why are most molecular helices right-handed?
9. Why do β-sheets tend to aggregate?
   • What is the driving force for β-sheet aggregation?
10. Why do many amyloid diseases form β-sheets?
    • Can you use amyloid β-sheets as materials?
11. Design a β-sheet motif that forms a well-ordered structure.

Part B: Protein Analysis and Visualization

In this part of the homework, you will be using online resources and 3D visualization software to answer questions about proteins. Pick any protein (from any organism) of your interest that has a 3D structure and answer the following questions:

1. Briefly describe the protein you selected and why you selected it.
2. Identify the amino acid sequence of your protein.
   • How long is it? What is the most frequent amino acid? You can use this Colab notebook to count the frequency of amino acids.
   • How many protein sequence homologs are there for your protein? Hint: Use Uniprot’s BLAST tool to search for homologs.
   • Does your protein belong to any protein family?
3. Identify the structure page of your protein in RCSB
   • When was the structure solved? Is it a good quality structure? Good quality structure is the one with good resolution. Smaller the better (Resolution: 2.70 Å)
   • Are there any other molecules in the solved structure apart from protein?
   • Does your protein belong to any structure classification family?
4. Open the structure of your protein in any 3D molecule visualization software:
   • PyMol Tutorial Here (hint: ChatGPT is good at PyMol commands)
   • Visualize the protein as “cartoon”, “ribbon” and “ball and stick”.
   • Color the protein by secondary structure. Does it have more helices or sheets?
   • Color the protein by residue type. What can you tell about the distribution of hydrophobic vs hydrophilic residues?
   • Visualize the surface of the protein. Does it have any “holes” (aka binding pockets)?

Part C. Using ML-Based Protein Design Tools

In this section, we will learn about the capabilities of modern protein AI models and test some of them in your chosen protein.

1. Copy the HTGAA_ProteinDesign2026.ipynb notebook and set up a colab instance with GPU.
2. Choose your favorite protein from the PDB.
3. We will now try multiple things in the three sections below; report each of these results in your homework writeup on your HTGAA website:

C1. Protein Language Modeling

1. Deep Mutational Scans
   1. Use ESM2 to generate an unsupervised deep mutational scan of your protein based on language model likelihoods.
   2. Can you explain any particular pattern? (choose a residue and a mutation that stands out)
   3. (Bonus) Find sequences for which we have experimental scans, and compare the prediction of the language model to experiment.
2. Latent Space Analysis
   1. Use the provided sequence dataset to embed proteins in reduced dimensionality.
   2. Analyze the different formed neighborhoods: do they approximate similar proteins?
   3. Place your protein in the resulting map and explain its position and similarity to its neighbors.

C2. Protein Folding

1. Fold your protein with ESMFold. Do the predicted coordinates match your original structure?
2. Try changing the sequence, first try some mutations, then large segments. Is your protein structure resilient to mutations?

C3. Protein Generation

1. Analyze the predicted sequence probabilities and compare the predicted sequence vs the original one.
2. Input this sequence into ESMFold and compare the predicted structure to your original.

Part D. Group Brainstorm on Bacteriophage Engineering

1. Find a group of ~3–4 students
2. Read through the Phage Reading material listed under “Reading & Resources” below.
3. Review the Bacteriophage Final Project Goals for engineering the L Protein:
   • Increased stability (easiest)
   • Higher titers (medium)
   • Higher toxicity of lysis protein (hard)
4. Brainstorm Session
   • Choose one or two main goals from the list that you think you can address computationally (e.g., “We’ll try to stabilize the lysis protein,” or “We’ll attempt to disrupt its interaction with E. coli DnaJ.”).
   • Write a 1-page proposal (bullet points or short paragraphs) describing:
     • Which tools/approaches from recitation you propose using (e.g., “Use Protein Language Models to do in silico mutagenesis, then AlphaFold-Multimer to check complexes.”).
     • Why do you think those tools might help solve your chosen sub-problem?
     • Name one or two potential pitfalls (e.g., “We lack enough training data on phage–bacteria interactions.”).
     • Include a schematic of your pipeline.
   • This resource may be useful: HTGAA Protein Engineering Tools
5. Each individually put your plan on your HTGAA website
   • Include your group’s short plan for engineering a bacteriophage

Week 5 — Protein Design Part II

This week we learn how cutting-edge AI and protein language models are used to design functional proteins and peptides “in silico”.

Homework — DUE BY START OF MAR 10 LECTURE

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Week 6 — Genetic Circuits Part I: Assembly Technologies

This week we learn core molecular biology tools and techniques for processing and assembling DNA, including PCR and Gibson Assembly.

Homework — DUE BY START OF MAR 17 LECTURE

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Week 7 — Genetic Circuits Part II: Neuromorphic Circuits

This week covers neuromorphic genetic circuits, showing how engineered gene networks can implement neural-network “perceptron”-like computation and learning.

Homework — DUE BY Mar 24 2PM ET

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Week 9 — Cell-Free Systems

This week introduces synthesis of proteins using cellular machinery outside of a cell.

Homework — DUE BY START OF Apr 7 LECTURE

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Week 10 — Advanced Imaging & Measurement Technology

This lecture presents a range of advanced technologies to do precision measurement of proteins at atomic scales, characterizing chemical composition, and detecting protein sequence and structure.

Homework — DUE BY START OF Apr 14 LECTURE

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Week 11 — Bioproduction & Cloud Labs

This week examines how modern bioproduction pipelines, from strain engineering to fermentation and downstream processing, are increasingly designed, executed, and optimized through cloud lab platforms and automation — enabling remote, high-throughput, and reproducible synthetic biology at industrial scale.

Homework — DUE BY START OF APR 21 LECTURE

(TBD)

Week 12 — Building Genomes

This week focuses on designing, synthesizing, and editing whole genomes, from minimal cells to refactored microbes and synthetic chromosomes.

Homework — DUE BY START OF APR 28 LECTURE

(TBD)

Week 13 — Biodesign & Engineered Living Materials

This week covers designing, programming, and fabricating engineered living materials — such as self-healing concretes, adaptive biofilms, and responsive biomaterials — by integrating genetic circuit design, materials science, and bioprocess engineering.

Homework: Work on your Final Project
Present it May 12 (MIT/Harvard) or May 13 (Committed Listeners)

Week 14 — Bio Design & Bio Fabrication

We wrap up the term looking towards a future of Bio-Design and Bio-Fabrication.

Homework: Finish your Final Project
Present it May 12 (MIT/Harvard) or May 13 (Committed Listeners)

Individual Final Project

For your Individual Final Project, write up your project and results on your HTGAA webpage following the guidelines below. You will present your project from that online writeup; presentations should be 6 minutes for MIT/Harvard students and 3 minutes for Global Committed Listeners, with 1-2 minutes of questions and discussion following.

Links: Links will be available later in the semester:

• (Signup sheets for a presentation slot)
• (A schedule of MIT/Harvard TA availability for lab work)
• (Signup sheet for MIT/Harvard Lab slots)

(View Full Screen)

Group Final Project

Phage Therapy

Phage therapy is the therapeutic use of bacteriophages to treat bacterial infections. Bacteriophages, or phages, are viruses that infect bacteria. They are highly specific, often infecting only a single strain of bacteria. Because of this specificity, phage therapy has potential advantages over traditional antibiotic treatment, which can kill beneficial bacteria along with the harmful ones. Phage therapy is seen as a solution to the problem of antibiotic resistance, which is becoming prevalent worldwide. At the current trend, in 26 years, the number of deaths attributed to antibiotic resistance are projected to become comparable with the number of deaths caused by cancer.

This project represents our HTGAA large-scale group research effort, where every participant has the opportunity to contribute to state-of-the-art research using advanced techniques. The potential impact of this work is significant, with the potential to make a real difference in people’s lives. For instance, consider the Patterson story, which highlights the transformative power of phage therapy. This Group Final Project is not just about academic advancement, but about making a tangible difference in the global fight against antibiotic resistance.

A famous example: Tom Patterson and Steffanie Strathdee’s story

As evidenced in the Patterson story, a significant challenge in phage therapy is the ability of bacteria to rapidly develop resistance to the phages. In Patterson’s case, the initial phage cocktail became ineffective after a few days. Consequently, another cocktail was developed and administered. Once again, it became ineffective after a short period. It was not until the third cocktail was introduced that the patient was finally cured. This highlights the need for continual monitoring and adaptation in phage therapy, reflecting the dynamic nature of bacterial resistance.

You can find a detailed introduction into bacteria, phages and phage therapy in our 2025 HTGAA Bootcamp Part 1

The Group Final Project

Despite the great advantages of phage therapy over conventional antibiotics, bacteriophages have a major limitation: Bacteria can develop ways to defend themselves against the phages and become resistant. But, in contrast to the static nature of antibiotics, phages have the power to evolve too. For billions of years, there has been an arms race between phages and bacteria. But now, with the development of new tools in synthetic biology such as protein engineering and the synthesis of new genomes harboring advantageous mutations, we can try to give the phages a head start. We attempt this by engineering their DNA or RNA, so they are prepared in case they encounter bacteria developing a resistance. This is the overall goal of the group final project. Specifically, we want to engineer the bacteriophage MS2 to be more prepared and more efficient in killing its host bacteria Escherichia coli (E. coli).

MS2 bacteriophage

MS2 bacteriophages infect E. coli bacteria with a high specificity. It is a very small virus consisting of coat proteins, a maturation protein and genetic material. They infect bacteria by attaching to the F-pilin protein on the host cell membrane and entering the cell. Once inside, the viral RNA acts as a messenger for phage protein production. All proteins for virion assembly are translated and the virus RNA is replicated. Next, new viruses form by assembling the coat and maturation proteins and encapsulating the virus RNA. Finally, a lysis protein expressed from the viral RNA triggers bacterial lysis by causing cell wall breakdown, whereby the new phages are released into the environment to infect new bacteria.

Empowering MS2 in their fight against E. coli

To make phages stronger against their host and to prepare them against host resistance, we can introduce mutations into their genes to slightly change the structure and/or function of the encoded proteins. Four genes are present on the MS2 RNA: 1. the maturation protein (A), 2. the coat protein (coat), 3. the lysis protein (L) and 4. the replicase (rep). For this course, we focus on the L gene encoding the lysis protein.

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446614/

The exact function of the L protein is unknown, however, it is thought to form oligomers that can then integrate into the cell membrane to form pores, which ultimately lyse and kill the bacterial cell. This lysis protein is crucial for the phage to complete its life cycle and release new phages to infect more bacteria. Because of its importance, E. coli can try to intervene in the lysis protein production to stop the spread of the phages. For the translation and processing of the viral proteins, the phage heavily relies on the bacterial protein machinery. One example for such a protein is DnaJ, a chaperone responsible for proper protein folding, which has been shown to be important for MS2 lysis protein processing. E. coli can mutate this chaperone, preventing the lysis protein from interacting with DnaJ. This in turn causes the lysis protein to lose its function and stops MS2 from infecting more bacteria.

In this project, we want to engineer the lysis protein to increase the ability of MS2 to overcome potential E. coli resistance. We can attempt this by mutating the lysis protein to change its properties. Together, we aim for finding mutations that change the lysis protein one of the following ways: (1) an independence of lysis protein processing from DnaJ or other bacterial chaperones and (2) a faster or more efficient killing of E. coli to reduce the window in which the host can acquire resistance. In the course of this class, we will proceed through the following stages to create and test new MS2 phage mutants:

Stage 1: Engineer novel L protein mutants using protein design tools

Stage 2: Synthesize the L protein mutant gene via Twist

Stage 3: Clone the L protein mutant gene into a plasmid using Gibson Assembly

Stage 4: Test the L protein mutant’s structural integrity using the Nuclera system

Stage 5: Test the L protein in E. coli (details will follow)

With this group project, each student will have the opportunity to actively contribute to authentic scientific research that will advance scientific knowledge and could potentially have an impact on people’s lives.

In Depth Reading Material

• Identification MS2 lysis protein dependency on DnaJ
• Mutational analysis of the MS2 lysis protein L
• Characterization of the MS2 lysis protein properties
• Phage therapy: From biological mechanisms to future directions
• Phage-therapy-past,-present-and-future