Week 1: Principles and Practices
Synthetic Bioogy in Regenerative Medicine: Drug Delivery The convergence of biology and engineering offers innovative potential to address complex healthcare challenges. More specifically, regenerative medicine has advanced enormously through inspiration from nature. Nowadays, bioengineered materials can be adapted to mimic and integrate natural designs with intricate mechanisms found in living organisms, ecosystems, and evolutionary processes. The main goal is to develop new materials, devices, and systems that can restore and enhance tissue performance and function, leading to new therapeutic approaches. Several essential synthetic biology techniques are used toward this aim, such as genetic engineering, cellular reprogramming, cellular pathway engineering, CRISPR-Cas9, delivery systems, artificial cells and organs, stem cell engineering, biomechanics, and bioinformatics.
🤖 Opentrons Liquid-Handling Artwork 🧠 Project Overview This project transforms the Opentrons OT-2 liquid handling robot into a biological plotter. Using coordinate-based programming, the robot deposits fluorescent bacterial droplets onto an agar plate to form a structured artistic pattern.
Part A. Conceptual Questions Answer any NINE of the following questions from Shuguang Zhang: (i.e. you can select two to skip) How many molecules of amino acids do you take with a piece of 500 grams of meat? For this exercise, it is necessary to assume that 20% of the meat is protein; therefore, 500 g of meat contains 100 g of protein.
Week 5: Protein design part ii
Superoxide dismutase 1 (SOD1) is a cytosolic antioxidant enzyme that converts superoxide radicals into hydrogen peroxide and oxygen. In its native state, it forms a stable homodimer and binds copper and zinc. Mutations in SOD1 cause familial Amyotrophic Lateral Sclerosis (ALS). Among them, the A4V mutation (Alanine → Valine at residue 4) leads to one of the most aggressive forms of the disease. The mutation subtly destabilizes the N-terminus, perturbs folding energetics, and promotes toxic aggregation.
Week 6: Genetic Circuits Part I - Assembly Technologies
Molecular Biology: PCR, Cloning & Transformation 1. Phusion High-Fidelity PCR Master Mix Components Phusion Hot Start II DNA Polymerase — synthesizes new DNA strands; has 3’→5’ exonuclease (proofreading) activity to correct misincorporated bases, giving very high fidelity. dNTPs (dATP, dCTP, dGTP, dTTP) — deoxynucleotide triphosphates; the building blocks incorporated into the growing DNA strand. MgCl₂ (magnesium chloride) — essential cofactor; Mg²⁺ ions stabilize the enzyme-DNA-dNTP complex and are required for catalytic activity. Optimized reaction buffer — maintains correct pH and ionic environment for efficient polymerase activity and primer annealing. Hot-start antibody/aptamer — inhibits polymerase at room temperature to prevent non-specific amplification; releases the enzyme once the initial high-temperature denaturation step is reached. 2. Factors Determining Primer Annealing Temperature GC content — G-C pairs have 3 hydrogen bonds vs. 2 for A-T; higher GC → higher Tm → higher annealing temperature. Primer length — longer primers have higher Tm due to more base-pair contributions to stability. Self-complementarity — hairpins or primer dimers reduce effective annealing temperature. Salt/ion concentration — higher Mg²⁺ or monovalent cations stabilize the duplex, raising Tm. Additives (formamide, DMSO) — destabilize base pairing, lowering effective Tm; useful for GC-rich regions. Mismatches — imperfect complementarity (e.g., mutagenic primers) requires lower annealing temperature. 💡 Rule of thumb: set annealing temperature ~5°C below the calculated Tm of the primer pair.
The convergence of biology and engineering offers innovative potential to address complex healthcare challenges. More specifically, regenerative medicine has advanced enormously through inspiration from nature. Nowadays, bioengineered materials can be adapted to mimic and integrate natural designs with intricate mechanisms found in living organisms, ecosystems, and evolutionary processes. The main goal is to develop new materials, devices, and systems that can restore and enhance tissue performance and function, leading to new therapeutic approaches. Several essential synthetic biology techniques are used toward this aim, such as genetic engineering, cellular reprogramming, cellular pathway engineering, CRISPR-Cas9, delivery systems, artificial cells and organs, stem cell engineering, biomechanics, and bioinformatics.
One particularly important tool is delivery systems. A common strategy to target specific locations is the use of nanoparticles (NPs). Due to their size and biocompatibility, NPs can breach biological barriers, penetrate deep tissues, and release therapeutic agents in a precisely controlled manner. Moreover, delivery system carriers can be tailored to respond to different conditions such as pH and temperature, enhancing treatment effectiveness while reducing unintended side effects.
Numerous studies have implemented this technique to deliver viral vectors for gene therapies in diseases such as hemophilia A and glioblastoma, as well as non-viral vectors, including proteins such as vascular endothelial growth factor. One particularly interesting protein that could be carried using NPs is the damage suppressor protein (Dsup), a nucleosome-binding protein found in the tardigrade Ramazzottius varieornatus (a resilient invertebrate commonly known as a water bear). This protein has been shown to significantly improve cell survival and growth by protecting against extreme stress conditions, specifically oxidative stress (hydrogen peroxide, H₂O₂) and UV-C irradiation in HEK293 human cells (human embryonic kidney cells).
Given the possibility of tailoring release conditions in nanoparticle-based delivery systems and the potential of Dsup to prevent DNA damage, I propose to study the delivery of Dsup using different types of nanoparticles into fibroblasts, the main cells found in skin. The skin is the largest organ of the human body and performs multiple functions, including homeostatic regulation; prevention of percutaneous loss of fluid, electrolytes, and proteins; temperature maintenance; sensory perception; and immune surveillance. This approach could help prevent cellular damage in degenerative phenomenon such as skin aging, which do affect every single person worldwide.
Minimize Biological and Long-Term Risks
Ensure that nanoparticle carriers and delivered proteins do not induce genotoxicity, immune dysregulation, or unintended cellular adaptations.
Prevent long-term accumulation or persistence of nanoparticles in tissues.
Prevent Misuse or Dual-Use Risks
Avoid applications that could enable enhancement beyond therapeutic intent (e.g., extreme stress resistance for non-medical or military use).
Ensure that the technology is not repurposed for harmful or coercive applications.
Promote Responsible and Equitable Access
Ensure that benefits are not restricted to cosmetic or luxury applications while excluding broader public health needs.
Encourage transparency and public engagement regarding intended uses.
Mandatory Preclinical Risk Assessment Framework
Actors: Academic researchers, funding agencies, Institutional Review Boards (IRBs)
Purpose: Current research on nanoparticle-based protein delivery systems often prioritizes short-term efficacy and cytotoxicity. This action proposes expanding existing requirements to include standardized assessments of long-term genomic stability, epigenetic alterations, immune responses, and nanoparticle persistence prior to clinical translation.
Design:
Funding agencies require comprehensive long-term safety evaluations as a condition for grant approval.
Scientific journals mandate extended safety datasets for publication.
IRBs implement nanoparticle-specific risk assessment protocols during project approval.
Assumptions:
Long-term biological effects can be reasonably predicted using advanced in vitro and animal models.
Research institutions possess or can access the infrastructure needed for extended safety testing.
Risks of Failure & “Success”:
Failure: Increased regulatory requirements may slow innovation or disproportionately impact under-resourced laboratories.
Success Risk: Excessive standardization could discourage exploratory research or unconventional delivery strategies.
Use-Based Regulatory Classification of Nanoparticle Applications
Actors: Federal regulators, public health authorities, regulatory agencies
Purpose: Instead of regulating nanoparticle delivery systems solely based on their material composition, this action proposes classifying them according to intended use (therapeutic, preventive, cosmetic, or enhancement-related), allowing for proportional oversight.
Design:
Regulatory agencies establish distinct approval pathways based on application category.
Therapeutic and disease-prevention uses receive prioritized evaluation.
Enhancement-oriented or cosmetic applications are subject to stricter scrutiny or limitations.
Assumptions:
Clear and enforceable distinctions between therapeutic and enhancement uses can be maintained.
Developers will accurately disclose the intended use of their products.
Risks of Failure & “Success”:
Failure: Ambiguous classifications could create regulatory loopholes.
Success Risk: Over-restriction may incentivize unregulated or informal markets for enhancement applications.
Technical Safeguards Embedded in Delivery System Design
Actors: Bioengineers, biotechnology companies, translational researchers
Purpose: This action promotes the integration of safety and misuse-prevention mechanisms directly into nanoparticle delivery systems to reduce the risk of unintended or unethical applications.
Design:
Nanoparticles engineered to degrade rapidly outside specific tissue microenvironments.
Activation of therapeutic cargo dependent on cell-type-specific enzymes or physiological conditions.
Regulatory agencies offer expedited review pathways for designs incorporating built-in safeguards.
Assumptions:
High levels of biological specificity can be reliably engineered into delivery systems.
Added design complexity does not compromise therapeutic performance.
Risks of Failure & “Success”:
Failure: Biological variability may limit the effectiveness of technical safeguards.
Success Risk: Increased development costs could reduce accessibility, particularly in low-resource settings.
| Does the option: | Option 1 | Option 2 | Option 3 |
|---|---|---|---|
| Enhance Biosecurity | |||
| • By preventing incidents | 1 | 2 | 1 |
| • By helping respond | 2 | 1 | 3 |
| Foster Lab Safety | |||
| • By preventing incidents | 1 | 2 | 2 |
| • By helping respond | 1 | 2 | 3 |
| Protect the environment | |||
| • By preventing incidents | 2 | 2 | 1 |
| • By helping respond | 2 | 3 | 2 |
| Other considerations | |||
| • Minimizing costs and burdens to stakeholders | 3 | 1 | 2 |
| • Feasibility | 2 | 1 | 2 |
| • Not impede research | 3 | 1 | 2 |
| • Promote constructive applications | 1 | 1 | 1 |
Based on the scoring, I recommend prioritizing Option 1 (Mandatory Preclinical Risk Assessment) and Option 2 (Use-Based Regulatory Classification), with selective use of Option 3 (Embedded Technical Safeguards) in later-stage applications.
Option 1 provides the strongest protection against biological and laboratory risks, which is essential given uncertainties around the long-term effects of nanoparticle-mediated protein delivery. Although it increases research burden, this trade-off is justified at early stages where prevention is most effective.
Option 2 adds proportional, use-based oversight that is highly feasible and minimizes unnecessary constraints on innovation, particularly during translation and commercialization.
Option 3 should be incentivized rather than required, as embedded safeguards reduce misuse risks but may increase complexity and costs. Together, this layered approach balances safety, feasibility, and responsible innovation.
Springer Reference. (2016). Nanoparticles in drug delivery. In Encyclopedia of Nanotechnology. Springer.
https://doi.org/10.1007/978-3-662-47398-6_4
Springer. (2024). Advances in regenerative medicine and tissue engineering. In Handbook of Regenerative Medicine (pp. 521–525). Springer.
https://doi.org/10.1007/978-3-031-87744-5
Madkour, L. H., et al. (2021). Nanoparticles and their biomedical applications. Biology, 10(10), 970.
https://doi.org/10.3390/biology10100970
World Health Organization. (2022). Global guidance framework for the responsible use of the life sciences: Mitigating biorisks and governing dual-use research. WHO.
https://iris.who.int/handle/10665/362313
Church, G. M., & Baker, D. (2024). Protein design meets biosecurity. Science, 383(6679), eado1671.
https://doi.org/10.1126/science.ado1671
This project transforms the Opentrons OT-2 liquid handling robot into a biological plotter.
Using coordinate-based programming, the robot deposits fluorescent bacterial droplets onto an agar plate to form a structured artistic pattern.
The objective was to:
The selected design is inspired by the Yin-Yang symbol, representing:
Robot: Opentrons OT-2
Pipette: P20 Single Channel Gen2
Agar Plate: Custom HTGAA agar plate
Fluorescent Strains:
| Color | Fluorescent Marker |
|---|---|
| Green | mClover3 |
| Orange | Azurite |
Each coordinate corresponds to a 1.5 µL droplet deposited at a specific XY location relative to the plate center.
Below is the complete script used for the Opentrons simulation and execution.
Before running on the real robot, the protocol was validated using the Opentrons simulator.
The robot successfully deposited bacteria following the coordinate map. After incubation, bacterial growth revealed the intended image on the agar plate.
This demonstrates that liquid-handling robots can perform microscale spatial biofabrication, a technique related to:
Figure. Overview of the HYDRA method.
A liquid-handling robot dispenses and re-aspirates hydrogel precursor solution to leave a micrometer-thin planar hydrogel inside multi-well plates, enabling biomimetic cell culture compatible with high-throughput drug screening and imaging.
HYDRA (HYDrogels by Robotic liquid handling Automation) presents a scalable and automated method to fabricate thin, uniform hydrogel layers inside standard multi-well plates used for high-throughput screening (HTS).
Traditional cell culture relies on rigid plastic or glass substrates, which poorly mimic the mechanical environment of real human tissues. This lack of physiological relevance contributes to high drug failure rates in clinical trials.
HYDRA addresses this issue by introducing planar hydrogel coatings (10–50 µm thick) that better replicate tissue stiffness while remaining fully compatible with imaging-based screening systems.
The key challenge solved by the study is meniscus formation in small wells, which normally leads to uneven gel surfaces and poor imaging quality. The authors developed a robotic workflow that deposits and re-aspirates hydrogel precursor solution to leave behind a controlled, micrometer-thin film.
The system was validated using:
Results demonstrated:
Conclusion: HYDRA enables more biomimetic and predictive drug testing without requiring new laboratory infrastructure.
The innovation of the paper lies in combining biomaterials + robotics.
An Opentrons OT-2 liquid-handling robot was programmed to:
Using Opentrons transforms hydrogel fabrication into a standardized, scalable microfabrication process:
Instead of using the robot as a simple pipetting tool, HYDRA turns it into a biomaterial fabrication platform — enabling physiologically relevant substrates directly inside standard drug screening plates.
This project proposes to automate the screening and characterization of nanoparticle delivery systems for the delivery of the damage suppressor protein (Dsup) — a nucleosome-binding protein from Ramazzottius varieornatus (tardigrade). Dsup has been demonstrated to protect mammalian cells from oxidative stress and UV-induced DNA damage.
The goal is to determine which nanoparticle formulation most effectively delivers Dsup into human dermal fibroblasts, improving resistance to oxidative stress (H₂O₂ exposure). The long-term application is skin regeneration and anti-aging therapies.
Automation tools (Opentrons OT-2 + cloud lab integration) will be used to:
Prepare nanoparticle formulations
Perform controlled protein loading
Treat fibroblast cultures
Apply oxidative stress
Perform viability assays
Collect quantitative data
flowchart TD
START[Start Protocol]
START --> LOAD[Load Labware & Tips]
LOAD --> PREP_NP[Prepare Nanoparticles]
PREP_NP --> LOAD_DSUP[Load Dsup Protein]
LOAD_DSUP --> INCUBATE1[Incubate Protein Loading]
INCUBATE1 --> SEED[Seed Fibroblasts]
SEED --> ADD_TREATMENT[Add NP-Dsup Treatment]
ADD_TREATMENT --> INCUBATE2[24h Incubation]
INCUBATE2 --> ADD_STRESS[Add H2O2]
ADD_STRESS --> INCUBATE3[Stress Incubation]
INCUBATE3 --> ADD_ASSAY[Add Viability Reagent]
ADD_ASSAY --> READ[Transfer to Reading Plate]
READ --> END[Export Data]| Instrument | Function |
|---|---|
| Echo | Transfers precise nanoliter volumes of Dsup protein and reagents |
| Bravo | Dispenses cell-free protein expression (CFPS) reagents into plates |
| Multiflo | Adds media, buffers, and treatment solutions across wells |
| Inheco | Provides controlled temperature incubation during reactions |
| PlateLoc | Seals microplates to prevent contamination and evaporation |
| PHERAstar | Measures fluorescence output for cell viability and protein activity |
How many molecules of amino acids do you take with a piece of 500 grams of meat?
For this exercise, it is necessary to assume that 20% of the meat is protein; therefore, 500 g of meat contains 100 g of protein.
Now, an amino acid has an average mass of 100 Daltons, and since
1 Dalton = 1 g/mol,
then 100 Daltons = 100 g/mol.
$$ \frac{100 g}{100 g/mol} \times 6.022 \times 10^{23} = 6.022 \times 10^{23} $$
Why do humans eat beef but do not become a cow, eat fish but do not become fish?
Humans do not become cows or fish when eating them because dietary proteins are digested into individual amino acids and small peptides in the gastrointestinal tract. The original three-dimensional structure and biological function of these proteins are destroyed during digestion. These amino acids are then reused by our cells to synthesize new proteins according to our own DNA sequence. Biological identity is determined by genetic information encoded in DNA, not by the proteins we consume. Therefore, although we obtain amino acids from beef or fish, we use them to build human proteins, not cow or fish proteins.
Why are there only 20 natural amino acids?
Can you make other non-natural amino acids? Design some new amino acids.
Where did amino acids come from before enzymes that make them, and before life started?
If you make an α-helix using D-amino acids, what handedness (right or left) would you expect?
Can you discover additional helices in proteins?
Why are most molecular helices right-handed?
Why do β-sheets tend to aggregate?
What is the driving force for β-sheet aggregation?
Why do many amyloid diseases form β-sheets?
Can you use amyloid β-sheets as materials?
Design a β-sheet motif that forms a well-ordered structure.
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:
Identify the amino acid sequence of your protein.
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) The Structures of Native Human Thymidine Phosphorylase and in Complex with 5-Iodouracil was solved in 2009 by the Department of Biology and Biochemistry, University of Bath with i high resolution of just 2.50 Å.
Are there any other molecules in the solved structure apart from protein? Yes. Besides the protein, the structure contains the small molecule 5-iodouracil (IUR), which is bound to the active site of thymidine phosphorylase. represented by C4 H3 I N2 O2
Does your protein belong to any structure classification family? Yes. The protein belongs to the nucleoside phosphorylase/phosphoribosyltransferase structural superfamily.
Superoxide dismutase 1 (SOD1) is a cytosolic antioxidant enzyme that converts superoxide radicals into hydrogen peroxide and oxygen. In its native state, it forms a stable homodimer and binds copper and zinc.
Mutations in SOD1 cause familial Amyotrophic Lateral Sclerosis (ALS). Among them, the A4V mutation (Alanine → Valine at residue 4) leads to one of the most aggressive forms of the disease. The mutation subtly destabilizes the N-terminus, perturbs folding energetics, and promotes toxic aggregation.
Your challenge:
💡 Rule of thumb: set annealing temperature ~5°C below the calculated Tm of the primer pair.
| Feature | PCR | Restriction Enzyme Digest |
|---|---|---|
| Output | Exponential amplification of a specific fragment | Cuts existing DNA at defined recognition sequences |
| Input | Template DNA + specific primers | Plasmid/genomic DNA + enzyme(s) |
| Sequence specificity | Defined by primer design | Defined by enzyme recognition site |
| End type | Blunt (Phusion) or designed overhangs | Blunt or sticky ends (enzyme-dependent) |
| Error risk | Possible polymerase errors | No replication errors; cuts are precise |
| Fragment size control | Any size; controlled by primer placement | Limited to where recognition sites naturally occur |
| Speed | ~1–3 hours | ~1 hour |
Gibson assembly requires 20–40 bp homologous overlaps between adjacent fragments.
Golden Gate Assembly is a scarless, one-pot DNA assembly method using Type IIS restriction enzymes (e.g., BsaI, Esp3I) and DNA ligase.