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

Class Assignment

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.).
   • Purpose: What is done now and what changes are you proposing?
   • Design: What is needed to make it “work”? (including the actor(s) involved - who must opt-in, fund, approve, or implement, etc)
   • Assumptions: What could you have wrong (incorrect assumptions, uncertainties)?
   • 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:

Your context:

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.

Weekly Assignment

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.

Final Project Assignment

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.

Before Next Class

• Open a Personal Notion Page (as covered in Recitation) and submit public link to your notion page to this Google Form.
• Complete the Principles and Practices Assignment (as shown above)

Specifically for In-Person Students at MIT/Harvard

• Lab Training (Failure to do so will jeopardize your acceptance)
  • 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)

Reading & Resources

Lab-specific

• Unit conversion & significant figures — Crash Course
• ▶️ Intro to pipetting

Governance & ethics

• Protein Design Meets Biosecurity: A 2024 Science editorial by George Church and David Baker
• Synthetic Genomics: Options for Governance This is an older but useful report for thinking about a variety of options for the governance of biotechnology that inspired this week’s homework
• US Presidential Commission on the Study of Bioethical Issues Report: “New Directions: The Ethics of Synthetic Biology and Emerging Technologies” This report considers the ethics of biotechnology and other emerging technologies
• Global guidance framework for the responsible use of the life sciences: “Mitigating biorisks and governing dual-use research” This World Health Organization report was issued after working groups from 2020-2022 and engendered much debate
• Bold Goals for U.S. Biotechnology and Biomanufacturing: “Harnessing Research and Development to Further Societal Goals” This 2023 report from the U.S. White House was generated as the result of an Executive Order
• National Security Commission on Emerging Biotechnology: This U.S. Congressional Commission will produce its first “comprehensive” report at the end of 2024 but has an “interim” 2023 report posted now, and they are currently soliciting input to guide national policy regulating biotech
• iGEM 2020 Safety Hub: This page includes links to many useful resources including the WHO biosafety manual, the NIH guidelines and the CDC Biosafety in Microbial and Biomedical Laboratories Guide; additional information is available on the iGEM 2023 Responsibility page
• Handbook for Community Biology Spaces: A handbook co-developed by community biolobabs, designed as a living document that can be updated and expanded by the community over time
• DIYBio Ask a biosafety expert This page includes a portal where you can get your biosafety questions answered by professionals
• Synopsis of Biological Safety and Security Arrangements (Gronvall & Rozo, 2015): This report describes biosafety and biosecurity rules and regulations in many countries
• Rooftop Solar and the Four Levers of Social Change: A blog post from Ethan Zuckerman considering different types of ways of regulating behavior, adopted in part from Lawrence Lessig’s book: Code 2.0, and explored in the context of energy consumption and production

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Lysis protein DNA sequence

atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa

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Lysis protein DNA sequence
```
atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa
```

stop

Lysis protein DNA sequence `atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa`

stop

Lysis protein DNA sequence atggaaacccgattccctcagcaatcgcagcaaactccggcatctactaatagacgccggccattcaaacatgaggattacccatgtcgaagacaacaaagaagttcaactctttatgtattgatcttcctcgcgatctttctctcgaaatttaccaatcaattgcttctgtcgctactggaagcggtgatccgcacagtgacgactttacagcaattgcttacttaa

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Lysis protein DNA sequence with Codon-Optimization

`ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA`

Lysis protein DNA sequence with Codon-Optimization
ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA

stop

Lysis protein DNA sequence with Codon-Optimization

ATGGAAACCCGCTTTCCGCAGCAGAGCCAGCAGACCCCGGCGAGCACCAACCGCCGCCGCCCGTTCAAACATGAAGATTATCCGTGCCGTCGTCAGCAGCGCAGCAGCACCCTGTATGTGCTGATTTTTCTGGCGATTTTTCTGAGCAAATTCACCAACCAGCTGCTGCTGAGCCTGCTGGAAGCGGTGATTCGCACAGTGACGACCCTGCAGCAGCTGCTGACCTAA

stop

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

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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)

SECTION 1: ABSTRACT

1. Provide an abstract/summary for your project. (minimum 150 words)

• Self-contained description of the project
• Should contain a brief outline of:
  • Significance
  • Broad objectives
  • Hypotheses
  • Specific aims
  • Methods to be employed
• Use lay language (i.e., understandable by the general public) as much as possible

SECTION 2: BACKGROUND

Provide background information and research for your final project. This should describe the current state of knowledge related to your project and should include a critical evaluation of the literature that identifies the gap in knowledge that this project will fill. Explain the larger context of your project area. What are the challenges and obstacles related to your project? What problem(s) are you seeking to address?

Cite at least 2 peer-reviewed research papers. Include any figures or visuals to help assist in explaining the background of your project area.

(min. 2 paragraphs)

SECTION 3: VISION and IMPACT

3a. Introduce the vision and impact of your final project. (min. 1-2 paragraphs) What is the overall vision and goal of your project? How will the world be different if your project is successful? Include figures and visuals as appropriate.

• Examples of topics to discuss
  • How your project solves a pressing problem in the world
  • Importance of the problem it solves or the critical barrier(s) to progress in the field that the proposed project addresses
  • The ways in which it contributes to the larger society
  • How the proposed project will improve scientific knowledge, technical capability, and/or clinical practice in one or more broad fields
  • How the concepts, methods, technologies, treatments, services, or preventative interventions that drive this field will be changed if the proposed aims are achieved

3b. Describe how your project is innovative (min. 3 sentences)

• Examples of topics to discuss
  • Novel applications, usage, or development of theoretical concepts, approaches, methodologies, instrumentation, and/or interventions
  • How it challenges current theories, paradigms, or ways in which technology/biological tools are used
• How your project pushes the boundaries of synthetic biology

3c. Describe the bioethical considerations involved in your project. (min. 2 paragraphs)

• First paragraph: Include what ethical implications are involved in your project. Try to suggest ethical the principle(s) you may apply (e.g. non-maleficence, justice)?
• Second paragraph: Describe the measures that should be taken to ensure that your project is ethical (both in how the research is conducted and in its broader implications for society). You may wish to answer the following questions:
  • What action(s) do you propose?
  • What are potential unintended consequences of your proposed actions?
  • What could you have wrong (e.g., incorrect assumptions and uncertainties)?
  • What are alternatives to your proposed actions?

SECTION 4: PROJECT AIMS

Outline three aims of your final project (min. 3 sentences, at least one for each aim)

1. The first aim should be structured “The first aim of my final project is to [insert an achievable experimental goal that encompasses your project] by utilizing [insert protocols/tools/strategies you will use to achieve your goal]”
   • State or link any methods/experimental protocols/OpenTrons protocols/DNA or protein designs/protein design tools or models/Twist orders you will use
   • You will provide a detailed, step-by-step outline of how you will achieve your goal for the first aim in the experimental design portion of this assignment (i.e., in question 7)
     • Feel free to run your goal by a TA
2. The second aim should be a medium-term aim that is a follow-up to your first aim and focused on goals beyond this class, building toward your third, visionary aim
   • For example, your second aim may be to successfully execute a set of experimental protocols, solve a specific problem, or develop a specific technology building upon the goals of your first aim.
3. The third aim should be a visionary, long-term aim
   • Reveal how the larger goal of the project can be impactful
   • Examples: challenging an existing paradigm or clinical practice, addressing a critical barrier to progress in the field, describing how you envision a new technology to change how a certain type of research is conducted

SECTION 5: EXPERIMENTAL DESIGN

Share a detailed experimental plan for your final project. Include a timeline for each part of your experimental plan (i.e., how long you would expect each step in your final project to take). (min. 15 lines/sentences—a numbered list is acceptable)

• Include specific methods/tools/technologies/biological concepts for each part of the final project and analysis
• Include any details related to any DNA designs and explain your design choices
• This section will be used to determine whether the experiments are well designed, feasible, and likely to succeed in testing your hypothesis
• Often this section is broken into discrete tasks/sub-aims
• For each experiment and/or analysis, include a description of your expected results
• If possible, include figure(s) that visually shows a broad workflow of your project or a specific aspect of your experimental plan

SECTION 6: RESULTS

Share the experimental results of your project. Include figures, tables, graphs, etc. to represent your experimental work. What did you measure and what were the results of those measurements? These results can be from lab work, computational methods, etc.

Here is a non-exhaustive list of areas where you can provide results:

• Performing a PCR reaction using primers relevant to your final project
• Performing a Gibson assembly relevant to your final project
• Designing DNA relevant to your final project
• Creating and performing a cell-free assay related to your final project
• Creating and running a code to validate an aspect of your final project
• Developing a model or completing a computational analysis relevant to your project
• Designing DNA construct(s) that can express at least one gene of interest, ordering it (via Twist), and testing of the expression of the construct(s) (potentially using an OpenTrons robot)

In the event you were unable to run certain experiments, please share your expected results. What kind of data were you hoping to get and why? Show an example data set or plot for reference (this could be from the literature or something you draw or plot).

SECTION 7: DISCUSSION AND FUTURE WORK

7a. Discussion (2 paragraphs minimum). Interpret your results. What worked? What didn’t? What data met your expectations? What did not go as planned and why do you think it didn’t work?

Discuss any other aspects of your project here as well. What were foreseen and/or unforeseen challenges? What strategies did you employ, or will you employ, to overcome these challenges?

7b. Future Work (1 paragraph minimum).

What are the next immediate steps you would like to take? What is your short term future work? What is your medium and long term future work? How do these plans relate to your aims?

SECTION 8: TECHNIQUES, TOOLS, AND TECHNOLOGY

8. We discussed and practiced various techniques related to synthetic biology throughout the semester. Place a check next to the techniques relevant to your project.

• Foundational Lab Practices
  • Pipetting
  • Lab Safety
  • Bioethical Considerations (must check this box)
• DNA Skills & Analysis
  • DNA Gel Art
  • DNA Sequencing
  • DNA Editing (e.g., CRISPR)
  • DNA Construct Design
  • Restriction Enzyme Digestion
  • Gel Electrophoresis
  • DNA Purification from Gel
  • Databases (e.g., GenBank, NCBI, Ensembl, UCSC Genome Browser)
• Laboratory Automation
  • Opentrons
    • Creating Code for Laboratory Automation
    • Using Liquid Handling Robots (e.g., Opentrons)
• Protein Design
  • Protein Design
    1. Models and Notebooks
    2. Databases
    3. Tools
• BioProduction
  • Chassis Selection (e.g., DH5α, Bl21-DE3 for expression)
  • Registry of Standard Biological Parts
  • FreeGenes
  • Plasmid Preparation
  • Bacterial Culturing
  • Quality Control / Analysis
  • Bacterial Processing (e.g., Centrifugation, Lysis, DNA Purification)
• Cell-Free System
  • Cell-Free Reactions
  • Freeze-Dried Cell-Free Systems
  • NEB Express Kits
  • miniPCR Tools
• Gibson Assembly
  • Primer Design or Selection
  • PCR Reactions
  • Gibson Assembly
  • Other Cloning Methods (e.g., Restriction Enzyme Digestion or Gateway Cloning)
  • Creating Twist Order

9. Expand upon two techniques you checked in the previous question by describing how you would utilize those techniques in your final project. (min. 4 sentences

SECTION 9: ADDITIONAL INFORMATION

10a. List all references cited in this assignment (bullet-point list)

10b. Create a supply list and budget for your project (bullet-point list)

• What supplies, equipment, and budget is needed for your project to work?

Example Past Final Projects:

2022:

• Designing Antibodies with Language Models
• RGB Screen-Printing Fabric and Bio-Fibers
• Co-Creative Forms

2021:

• Microfluidic Covid-19 mRNA Vaccine Drug Delivery
• Bacterial Shading
• Immune-System-Friendly RNA Delivery

Example Past Class Webpages:

2022:

• Ido Calman
• Rosalie Lin
• Hyun Woo Park (Hyun Parke)

2021:

• Danny Chen
• Laura Maria Gonzalez
• Dominika Wawrzyniak
• Danny Chen
• Laura Maria Gonzalez
• Dominika Wawrzyniak

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 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