Week 1 HW: Principles and Practices

1. First, describe a biological engineering application or tool you want to develop and why.

I want to develop a 3D Bio-Art Platform that merges biological growth with interactive synthetic biology. The idea is to use 3D-printed molds and structured agar media to create “living sculptures” that don’t just sit there but actually “feel” and react.

The sculpture uses a quorum sensing circuit to create organic, emergent color gradients as the bacteria colonize the 3D agar structure. However, by engineering the bacteria with inducible promoters sensitive to microcurrents, heat, or other factors, the sculpture reacts to human and environmental touch. When you touch a specific plate, the bacteria trigger a rapid flash of bioluminescence or a sharp color change. It’s a very solarpunk vision where the artwork is a living, sensing entity that bridges the gap between autonomous growth and intentional human interaction.

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.

Some of the main goals include the following:

A. Preventing Malicious Use & Biological Escape (Biosecurity: To ensure that the bacteria used in the sculptures cannot be extracted and repurposed or survive outside the controlled art environment. This could be achieved with the help of:

1. An Intrinsic Biological Lock: Implementing a strategy where the metabolic reagents and the bacterial chassis are only viable inside a specific chemical or mechanical environment of the 3D bio-art sculpture.

2. Genetic Safeguards: Using “kill switches” so the organisms are biologically incapable of surviving in the local ecosystem if the sculpture is broken, archived, or discarded.

3. Access Control & Registry: Establishing a “Bio-Art registry” where any high-expression or highly interactive strain is registered and tracked from the lab to the gallery or art exposition.

B. End User Safety & Interaction Reliability (Biosafety): To guarantee that the interaction between the public and the “living touch” interface is 100% safe, reliable and follows predictable patterns. This could be achieved with the help of:

1. Interaction Safety Protocols: Establishing clear “bio-etiquette” protocols and adding physical boundaries to prevent accidental ingestion, skin irritation from undesired contact, or environmental transfer during public exhibitions. Also, establishing risk protocols and measures for any accidents or incidents that could happen.

2. Contamination Control: Implementing a strategy to ensure that the emergent bacteria patterns are not contaminated by other wild-type bacteria from the users’ hands, which could ruin the artistic expression, 3D bio-art sculpture, and the biosafety protocols.

3. Real-time Stability Monitoring: Integrating “self-reporting” circuits and sensors where bacteria change to a “warning color” (like a bright red or yellow) if the population begins to mutate or if the containment is failing.

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

Action 1: Multi-Layered Kill Switches (Technical Strategy that can be applied through international organization like WHO, ASM, etc)

1. Purpose: Currently, containment is mostly physical. In this strategy, all interactive bio-art must use a “dead-end” genetic design.
2. Design: Using nutritionally dependent strains that require a synthetic, non-canonical nutrient embedded in the agar. Without this “artificial food,” the bacteria degrades immediately.
3. Assumptions: We assume that horizontal gene transfer in the environment won’t provide the bacteria a way to bypass this dependency.
4. Risks: A “success” might make the biology too fragile for long exhibitions but in a controlled manner, while a failure would be the organism finding a natural substitute for the synthetic nutrient, which could lead to unwavering growth.

Action 2: Public Interaction “Bio-Etiquette” Certification (New Requirement that is applied by the responsible company)

1. Purpose: To change how the public views OGM interaction from “dangerous” or “uncertain” to “responsible” and “reliable.”
2. Design: Any gallery exhibiting the 3d bio-art sculptures must implement a mandatory hand-sanitizing and briefing station. The actors here are the gallery owner and the artist.
3. Assumptions: We assume that the public will follow all instructions and not try to “vandalize” the sculpture by introducing outside contaminants.
4. Risks: Success creates a safe, educated public; failure is a “success” where the art becomes so popular that the safety protocols are ignored due to high traffic.

Action 3: Peer-Led Biosecurity Audit (Community Strategy that involves the public and synbio community, the artists and the responsible company)

1. Purpose: To move away from slow federal oversight and use the agility of the SynBio community locally and globally.

2. Design: A “Safety Buddy” system where a fellow scientist must audit the genetic circuits and the physical mold design before it leaves the lab.

3. Assumptions: We assume peers will be rigorous and not just let their friends’ projects go on without revising them.

4. Risks: Success builds a strong self-regulating culture. Failure is a lapse in judgment that leads to a public health scare, potentially getting bio-art banned or detained.

5. 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.

Based on the scored framework, I recommend that we prioritize action 1 (Technical Multi-Layered Kill Switches) as the foundation, supported by action 3 (Community Peer Led Biosecurity Audit).

The technical multi-layered kill switches are the only way to ensure the biology is ethical by design; if the bacteria can’t survive outside the mold, the “risk” is effectively zero. However, I’m trading off some technical simplicity for absolute peace of mind. On the other hand, the peer led biosecurity audit is important because it builds the “social tissue” of responsibility among us students. We don’t need more laws; we need better engineers and technicians who check each other’s work. Lastly, my biggest uncertainty is the mutation rate of the kill switches, which is why the community audit must be a recurring process, with constant feedback loops and not a one-time thing.

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.

This project made me realize that when we make biology “interactive” and “eye-catching,” we might lower people’s guard. However, a concern that arose was about the ethical autonomy of the biological parts of 3d bio-art: are we just “enslaving” these bacteria for a 3-second glow? Or are we letting them decide what is best for them? By using Action 3, we ensure that as artists and scientists, we are also managers of the life we modify, treating it with the respect and conscience it deserves.

Week 2 HW: DNA read, write & edit

Homework 02

Part 1: Benchling & In-silico Gel Art

• I successfully made a Benchling account and imported 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.

For my in-silico Gel Art I wanted to initially make a star! Sadly, after using Ronan’s website to visualize my idea, I realized that it would be a bit complicated using the listed Restriction Enzymes.

So, I ended up making some tulips instead! You can check out my design on Ronan’s website too!

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

I skipped this one since I do not have Lab access.

Part 3: DNA Design Challenge

3.1. Choose your protein.

I have chosen the Chitinase enzyme from the bacterium Bacillus thuringiensis (NCBI Accession: WCH14858.1).

I found this protein interesting because of its potential in environmental conservation and biotechnology. This enzyme is capable of degrading chitin, which is a primary component of fungal cell walls and insect exoskeletons. Based on the literature, the chitinase protein is particularly efficient due to its modular structure, which typically includes a catalytic domain and chitin-binding domains that enhance its hydrolytic activity (1). Because of that, this protein becomes a very powerful tool for biological control: it can act synergistically with Cry proteins to perforate the peritrophic matrix of insect pests, increasing the efficiency of biopesticides. Additionally, I selected this specific protein because Bacillus thuringiensis is a safe organism to handle in a Level 1 biosafety laboratory (BSL-1), making it a practical and efficient candidate for recombinant protein production in E. coli.

>WCH14858.1 chitinase [Bacillus thuringiensis] MLNKFKFFCCILVMFLLLPLSPFQAQAANNLGSKLLVGYWHNFDNGTGIIKLKDVSPKWDVINVSFGETGGDRSTVEFSPVYGTDAEFKSDISYLKSKGKKIVLSIGGQNGVVLLPDNAAKDRFINSIQSLIDKYGFDGIDIDLESGIYLNGNDTNFKNPTTPQIVNLISAIRTISDHYGPDFLLSMAPETAYVQGGYSAYGSIWGAYLPIIYGVKDKLTYIHVQHYNAGSGIGMDGNNYNQGTADYEVAMADMLLHGFPVGGNANNIFPALRSDQVMIGLPAAPAAAPSGGYISPTEMKKALNYIIKGVPFGGKYKLSNQSGYPAFRGLMSWSINWDAKNNFEFSSNYRTYFDGLSLQK

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

To determine the nucleotide sequence that corresponds to the chitinase protein I went to the original Bacillus thuringiensis genome.

[Original sequence of Bacillus thuringiensis from its genome Bacillus thuringiensis - Nucleotide - NCBI]

Chitinase protein DNA sequence
atgttaaacaagttcaaatttttttgttgtattttagtaatgttcttacttctaccgttatcccctttccaagcacaagcagcaaacaatttaggttcaaaattactcgttggatactggcataattttgataacggtactggcattattaaattaaaagacgtttcaccaaaatgggatgtaatcaatgtatcttttggtgaaactggtggtgatcgttccactgttgaattttctcctgtgtatggtacagatgcagaattcaaatcagatatttcttatttaaaaagtaaaggaaagaaaatagttctttcaataggtggacaaaatggggtcgttttacttcctgacaatgccgctaaggatcgttttattaattccatacaatctctgatcgataaatacggttttgacggaatagatattgaccttgaatcaggtatttacttaaacggaaatgacactaacttcaaaaacccaactacccctcaaatcgtaaatcttatttcagctattcgaacaatctcagatcattatggtccagattttctattaagcatggcccctgaaacagcttatgttcaaggcggatatagcgcatatggaagcatatggggtgcatatttaccaattatttatggagtgaaagataaactaacatacattcacgttcaacactacaacgctggtagcgggattggaatggacggtaataactacaatcaaggtactgcagactacgaggtcgctatggcagatatgctcttacatggttttcctgtaggtggtaatgcaaataacattttcccagctcttcgttcagatcaagtcatgattgggcttccagcagcaccagcggcagctccaagtggtggatacatttcgccaactgaaatgaaaaaagctttaaattatatcattaaaggagttccattcggaggaaagtataaactttctaaccagagtggctatcctgcattccgcggcctaatgtcttggtctattaattgggatgcaaaaaacaactttgaattctctagtaactatagaacatattttgatggtctttccttgcaaaaataa

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?

I need to optimize codon usage because, although the genetic code is redundant, different organisms have distinct ‘codon biases.’ Since I am using a sequence from Bacillus thuringiensis, I have optimized it for Escherichia coli K-12 using Benchling’s Codon Optimization tool to ensure that the host cell can translate it efficiently. I chose the K-12 strain specifically because it is the gold standard in synthetic biology laboratories since it is a safe, non-pathogenic, and well-characterized model that guarantees reliable folding for my chitinase enzyme.

Chitinase protein DNA sequence Codon-Optimization
ATGCTGAACAAATTTAAATTTTTTTGCTGCATTCTGGTGATGTTTCTGCTGCTGCCGCTGAGCCCGTTTCAGGCGCAGGCGGCGAACAACCTGGGCAGCAAACTGCTGGTGGGCTATTGGCATAACTTTGATAACGGCACCGGCATTATTAAACTGAAAGATGTGAGCCCGAAATGGGATGTGATTAACGTGAGCTTTGGCGAAACCGGCGGCGATCGCAGCACCGTGGAATTTAGCCCGGTGTATGGCACCGATGCGGAATTTAAAAGCGATATTAGCTATCTGAAAAGCAAAGGCAAAAAAATTGTGCTGAGCATTGGCGGCCAGAACGGCGTGGTGCTGCTGCCGGATAACGCGGCGAAAGATCGCTTTATTAACAGCATTCAGAGCCTGATTGATAAATATGGCTTTGATGGCATTGATATTGATCTGGAAAGCGGCATTTATCTGAACGGCAACGATACCAACTTTAAAAACCCGACCACCCCGCAGATTGTGAACCTGATTAGCGCGATTCGCACCATTAGCGATCATTATGGCCCGGATTTTCTGCTGAGCATGGCGCCGGAAACCGCGTATGTGCAGGGCGGCTATAGCGCGTATGGCAGCATTTGGGGCGCGTATCTGCCGATTATTTATGGCGTGAAAGATAAACTGACCTATATTCATGTGCAGCATTATAACGCGGGCAGCGGCATTGGCATGGATGGCAACAACTATAACCAGGGCACCGCGGATTATGAAGTGGCGATGGCGGATATGCTGCTGCATGGCTTTCCGGTGGGCGGCAACGCGAACAACATTTTTCCGGCGCTGCGCAGCGATCAGGTGATGATTGGCCTGCCGGCGGCGCCGGCGGCGGCGCCGAGCGGCGGCTATATTAGCCCGACCGAAATGAAAAAAGCGCTGAACTATATTATTAAAGGCGTGCCGTTTGGCGGCAAATATAAACTGAGCAACCAGAGCGGCTATCCGGCGTTTCGCGGCCTGATGAGCTGGAGCATTAACTGGGATGCGAAAAACAACTTTGAATTTAGCAGCAACTATCGCACCTATTTTGATGGCCTGAGCCTGCAGAAATAA

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.

To produce chitinase from my designed sequence, I can use either cell-dependent or cell-free methods. In a cell-dependent approach, I would insert the DNA into a host like E. coli K-12, where the cell’s own machinery handles the work: RNA polymerase transcribes the DNA into mRNA, and then ribosomes translate that message into the final enzyme. On the other hand, cell-free protein synthesis allows me to skip the living cell entirely by using just the necessary biological “parts” (like enzymes and ribosomes) in a tube. This last approach is a much faster way to prototype the protein without keeping bacteria alive, although I really have a space in my heart for bacterial cultures.

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.

From what I’ve understood, a single gene can produce different proteins through mechanisms like alternative splicing, where the cell mixes and matches different sections of the message (exons) to create several versions of a protein from the same DNA template. In bacteria like Bacillus thuringiensis, they also use polycistronic operons, which group several related genes under a single promoter. This allows the bacteria to produce a whole set of coordinated enzymes all at once.

2. Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!!

Part 4: Prepare a Twist DNA Synthesis Order

4.1. Create a Twist account and a Benchling account

4.2. Build Your DNA Insert Sequence

I’ll make a sequence that will make E. coli glow fluorescent blue under UV light by always expressing sfBFP (a blue fluorescent protein):

Screenshot of the creation of the sfBFP sequence in Benchling

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).

Screenshot of the whole sequence with its annotations!

• Promoter (BBa_J23106): TTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGC
• RBS (BBa_B0034 with spacers for optimal expression): CATTAAAGAGGAGAAAGGTACC
• Start Codon: ATG
• Coding Sequence (Codon optimized DNA sfBFP): ATGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGCGCGGCGAAGGCGAAGGCGATGCGACCAACGGCAAACTGACCCTGAAATTTATTTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGACCCATGGCGTGCAGTGCTTTAGCCGCTATCCGGATCATATGAAACGCCATGATTTTTTTAAAAGCGCGATGCCGGAAGGCTATGTGCAGGAACGCACCATTAGCTTTAAAGATGATGGCACCTATAAAACCCGCGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTTTAACAGCCATAACGTGTATATTACCGCGGATAAACAGAAAAACGGCATTAAAGCGAACTTTAAAATTCGCCATAACGTGGAAGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAACCATTATCTGAGCACCCAGAGCGTGCTGAGCAAAGATCCGAACGAAAAACGCGATCATATGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCATGGCATGGATGAACTGTATAAA
• 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 (BBa_B0015): CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA

Screenshot of the Linear map of Constitutive sfBFP DNA and here is the Benchling Link

SBOL of the Linear map of Constitutive sfBFP DNA.

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.

Screenshot of my uploaded sfBFP FASTA file in Twist

4.6. Choose Your Vector

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

Screenshot of sfBFP with pTwist Amp High Copy vector

My Twist Ready 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 want to sequence eDNA from river water samples collected at different points in different regions, especially near my hometown. Rivers collect DNA from fish, amphibians, and even terrestrial animals that drink from or live near the water. By sequencing the DNA, I can perform a biodiversity assessment to detect invasive species (like the trout in some Andean rivers) and/or monitor the presence of endangered amphibians without the need for traditional trapping methods.

(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?\

I would use Illumina (Next-Generation Sequencing) because its massive parallelization would allow me to read millions of sequences from hundreds of species in a single run, which is perfect for complex environmental samples (e.g., in rivers).

Also answer the following questions:

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

Illumina’s NGS is second-generation. That’s because it uses synthesis-based sequencing on a solid surface rather than reading single long molecules.

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

My input would be filtered river water DNA. Preparation involves metabarcoding (amplifying specific markers like 16S or COI) and adapter ligation to attach fragments to the flow cell.

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

Illumina’s NGS has many steps but these are the essential ones that make the work itself. First, DNA fragments are attached to a flow cell where they form dense clusters through bridge amplification to ensure the detection signal is strong enough. Next, fluorescently labeled nucleotides are added one by one, and a high-resolution camera records the specific color flash emitted as each base is incorporated into the strand. Finally, the software interprets these light patterns and decodes them into a digital DNA sequence through base calling. (2)

4. What is the output of your chosen sequencing technology?

The output of Illumina’s NGS is a FASTQ file containing millions of digital reads that identify the species present in the river samples. Once I get the file I can analyze it. with bioinformatics and get results.

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 like to synthesize a genetic biosensor designed to detect heavy metal contamination, such as mercury, in river water. By placing this circuit into a safe host like E. coli K-12, the bacteria could “glow” or change color when it senses toxins, acting as a real-time environmental monitor to help protect the river’s biodiversity.

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

I would love to use Twist Bioscience’s Silicon-based Synthesis to perform the DNA synthesis because of its incredible scalability and its promise of making DNA synthesis better and faster. (3)

Also answer the following questions:

1. What are the essential steps of your chosen sequencing methods?

The steps that Twist follows use silicon chips to print thousands of genes simultaneously, which significantly reduces costs and improves precision. First, the digital sequence is uploaded and ‘printed’ onto a silicon chip; using phosphoramidite chemistry, the machine builds thousands of short DNA strands, known as oligonucleotides, base by base. Second, these short oligos are harvested from the chip and gathered together. Finally, the fragments are enzymatically assembled to form the complete, full-length biosensor circuit, ensuring high precision and scalability.

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

The main limits are that very complex designs can significantly increase the turnaround time and the cost of production. Additionally, sequences with difficult content, such as high GC-rich regions, can lower the synthesis success rate.

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 like to edit the chitinase genes in native river bacteria to make them more efficient at degrading organic waste. This would help prevent fungal outbreaks and the accumulation of debris, keeping the river ecosystem balanced and clean in a natural way.

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

I would use CRISPR-Cas9 because it is the most precise, well-known, and easy-to-design tool for genome engineering in bacteria. The system works by using a guide RNA (gRNA) that leads the Cas9 nuclease to a specific target in the chitinase gene to create a cut. By providing a DNA repair template, I can then insert a more efficient version of the enzyme into the genome.

Also answer the following questions:

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

This technology edits DNA by acting like a pair of molecular scissors. It follows three main steps: first, the guide RNA identifies and binds to a specific target sequence in the genome. Second, the Cas9 nuclease creates a double-strand break at that exact location. Finally, the cell’s natural repair machinery goes and fixes the break; by providing a DNA repair template, the cell can be tricked into incorporating a new, more efficient chitinase sequence during this repair process.

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?

I would need to digitally design a specific gRNA that is perfectly complementary to the chitinase gene to avoid off-target cuts. Additionally, the required inputs for the experiment include the Cas9 protein (or a plasmid encoding it), the custom synthetic gRNA, a DNA donor template containing the desired edit, and the target bacterial cells that will be transformed with these components!

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

The biggest limitation of this method is the risk of off-target cuts, where the Cas9 might cut a similar DNA sequence elsewhere in the genome by mistake. Additionally, the efficiency of the edit depends a lot on the cell’s repair mechanism; in some bacteria, the rate of successful “homology-directed repair” can be low, meaning many cells might fail to incorporate the new gene correctly.

References

1. Martínez-Zavala, S. A., Barboza-Pérez, U. E., Hernández-Guzmán, G., Bideshi, D. K., & Barboza-Corona, J. E. (2020). Chitinases of Bacillus thuringiensis: Phylogeny, Modular Structure, and Applied Potentials. Frontiers in Microbiology, 10, 3032. https://doi.org/10.3389/fmicb.2019.03032
2. Next-Generation Sequencing (NGS) | Explore the technology.
3. Twist Bioscience. High quality gene synthesis - Twist Bioscience. Gene synthesis

Week 3 HW: Lab Automation

Homework

Assignment: Python Script for Opentrons Artwork

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

1. Generate an artistic design using the GUI at opentrons-art.rcdonovan.com.

For my first design I made a colorful butterfly! I first used the Opentrons art page to design it by using the upload image option. Initially the design

Here you can see the butterfly image that I uploaded and how it generates on the Opentrons art page side by side!

Then after the image upload, I decided to first move the design a bit lower and also change the colors. Lastly I added some fun details like stars and a heart.

Design process

Here’s the final design!

This is my design: a colorful butterfly! Made using the GUI. You can check it out by yourself here!

Initially, I made one artistic design on a circular petri dish, but after finding out you could make designs on a rectangular plate, I decided to try it out! I ended up making 2 more designs on rectangular plates.

This is my second design which is a readaptation of the colorful butterfly! Made using the GUI. You can check it out by yourself here!

This is my third design: an anomalocaris! Made using the GUI. You can check it out by yourself here!

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.

Here’s the Opentrons Lab Simulation in Google Colab for the first design. You can check it out by yourself here!

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.

Paper: Automation of protein crystallization scale-up via Opentrons-2 liquid handling.

This paper explores the use of the Opentrons OT-2 machine to automate protein crystallization! The researchers developed three Python scripts using the Opentrons Python module to control the robot for mixing and setting up 24-well sitting drop plates using model proteins like lysozyme and a periplasmic protein from Campylobacter jejuni.

The study achieved the desired scale-up goals after minimal trial and error. By automating the liquid handling, the researchers were able to test a wider range of crystallization conditions (reagents, concentrations, and pH) with higher reproducibility than manual pipetting. Although the setup time was around 35 to 40 minutes, it greatly reduces plate variability from person to person. This is a novel application because it makes high-quality structural biology workflows accessible and low-cost, allowing labs to screen protein conditions at a much higher throughput, which is essential for understanding protein function and drug design.

Reference: DeRoo, J. B., Jones, A. A., Slaughter, C. K., Ahr, T. W., Stroup, S. M., Thompson, G. B., & Snow, C. D. (2025). Automation of protein crystallization scaleup via Opentrons-2 liquid handling. SLAS Technology, 32, 100268. https://doi.org/10.1016/j.slast.2025.100268

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.

For instance, my first idea is an interactive 3D bio-art installation that translates a person’s biological data into a living and blooming sculpture. This idea uses genetically engineered bacteria to create a visual representation of a user’s unique microbial/DNA fingerprint. The process starts when a user interacts with a sensor that captures basic biological data, which is then processed by a script to assign specific colors using fluorescent proteins like GFP, RFP, and BFP. In this case, an Opentrons OT-2 acts as a high-precision bio-printer to deposit these living bio-inks into a 3D-printed scaffold made of agar or hydrogel, allowing the sculpture to grow and glow over time to reveal the user’s identity.

Additionally, I will need to design 3D-printed holders with micro-channels and a specialized needle adapter so the OT-2 can deposit the bacteria without breaking the hydrogel/agar structure. I will use capacitive touch sensors to generate the initial data that determines the bacterial/DNA distribution throughout the sculpture. Moreover, I plan to use cloud laboratories like Ginkgo Nebula to synthesize the custom DNA circuits needed to ensure the bacteria express the exact colors and intensity required for the piece.

Here’s a rough python pseudocode for this 3D sculpture idea.

from opentrons import protocol_api

# This script translates user data into a 3D bacterial pattern
def run(protocol: protocol_api.ProtocolContext):
    # Load the custom 3D printed lattice and the bio-inks (bacteria)
    sculpture_lattice = protocol.load_labware('custom_3d_lattice', '1')
    bio_inks = protocol.load_labware('opentrons_24_tuberack_eppendorf_1.5ml_safelock_snapcap', '2')
    p20 = protocol.load_instrument('p20_single_gen2', 'right')

    # Logic: If user data indicates Trait X, use Blue Fluorescent Protein
    user_trait = "high_diversity" # Example data from sensor
    
    if user_trait == "high_diversity":
        # Deposit Blue bacteria in the outer ring of the lattice
        for well in sculpture_lattice.rows()[0]:
            p20.pick_up_tip()
            p20.transfer(10, bio_inks['A1'], well, new_tip='never')
            p20.drop_tip()
            
    # Move in Z-axis to create the 3D effect
    p20.move_to(sculpture_lattice.wells()[0].top(z=10))

Final Project Ideas — DUE BY START OF FEB 24 LECTURE

As explained in this week’s recitation, add a slide in your Node’s section of this slide deck with an idea you have for an Individual Final Project. Be sure to put your name on your slide!

Here are my three individual final project ideas!

An interactive 3D bio-art sculpture

A river-sensing automated robot system

Chlorella vulgaris in silico optimization and automation