Week 1 HW: Principles and Practices

IDEA

Through art, design, biology, and apparel, I am interested in exploring how wearers can become more attuned to their identity by expressing their unique microbial genomes and ecosystems that make up their bodies through a bio-engineered second-skin-like textile. This project explores clothing as a custom, an expression, and a living interface that evolves with the body, offering an alternative to the rapid cycles of novelty and replacement characteristic of fashion today. The garment becomes a second skin that is not consumed or discarded, but lived with, every day, shifting gradually and meaningfully over time (National Institutes of Health).

I am drawn to the skin microbiome as a dynamic and richly complex ecosystem that continuously evolves in response to environmental stimuli, material contact, and everyday life. The skin absorbs and interacts with everything it encounters—from air and surfaces to residues such as microplastics—while remaining in constant dialogue with its own microflora. I envision a bespoke “second skin” generated through the bioengineering of an individual’s genomic profile and cutaneous microbiome, translated into algorithmically encoded, chromatically responsive pattern systems.

This work is inspired by Neri Oxman’s approach to material ecology, particularly her use of modularity, gradience, and unity to integrate biological processes directly into design (Marvin, J.). In this project, patterns within the material are speculated to evolve gradually as the wearer’s skin ecosystem undergoes microbial succession in response to environmental exposure, climate, and daily interaction. Bioengineered, non-pathogenic microbial consortia or biologically derived, metabolically active materials are embedded within the textile architecture, functioning as living intermediaries between the body and its environment. These systems respond to localized changes in the skin’s microenvironment, such as pH, moisture, temperature, and biochemical flux, through controlled shifts in pigmentation, pattern density, and material morphology (Atallah, C.).

Rather than interpreting or evaluating bodily states, the garment becomes a living material archive, reflecting a healthy, dynamic relationship among the wearer, their microbial ecosystem, and the world they move through. In this way, fashion becomes a site of ongoing exchange and evolution, replacing short-term satisfaction with a deeply personal, evolving form of embodiment.

In a previous exploratory project, “SECOND-SKIN”, I sampled and analysed skin-associated microbial communities from distinct anatomical regions, observing that different areas of the body host unique microbial ecologies. This work focused on translating the otherwise invisible microbiome of the human body into an expressive, artistic language.

In this project, I extend this inquiry by proposing a modular design and material system that reflects the evolution of an individual’s skin microbiome over time. The textile functions as a responsive biological interface or living biologically active material that registers changes in microbial composition, metabolic activity, or by-products present on the skin’s surface. These shifts are translated into gradual changes in colour, texture, or pattern, allowing the textile to act as a temporal record of the wearer’s ever-evolving microbial life.

Project “SECOND-SKIN” 2020

GOVERNANCE POLICY GOALS:

GOAL 1: Prevent Harm and Ensure Safety Because this textile directly interfaces with the body and incorporates living or biologically active components, governance must prioritise physical, biological, and environmental safety throughout design, experimentation, and use.

Sub Goal 1: Biological Safety Ensure the safety of the wearer’s skin and microbiome by regulating the use of non-pathogenic organisms, fully contained or controlled living systems, and biocompatible materials. Prevent adverse skin reactions and unintended microbial transfer during use, experimentation, and prolonged wear.

Sub Goal 2: Prevent Environmental Contamination Establish controlled experimental conditions, follow appropriate laboratory protocols, and ensure safe containment, deactivation, or disposal of biological components at the end of the material’s lifecycle to prevent unintended environmental release.

Sub Goal 3:Clarification and Communication Clearly communicate the material’s biological components, symbiotic role, and limitations to users and research participants. Avoid misclassification of the textile as a diagnostic, therapeutic, or medical technology by framing it explicitly as an expressive, cultural, and experiential material system.

GOAL 2: Protecting Autonomy, Consent and User Agency Working with microbiome-derived data and living materials raises ethical concerns related to bodily autonomy, consent, and ownership of biological information.

Sub Goal 1: Informed Consent Users should clearly understand what biological information is being used (e.g. abstracted microbiome composition), how it is translated into material form, and what is not being measured or inferred. Consent should be explicit, ongoing, and revisitable as the material evolves.

Sub Goal 2: Time, Control and Lifecycle Awareness Ensure users understand the material’s temporal nature, how it may evolve, change, or age and provide clear mechanisms for disengagement, pausing, or material retirement. Users should be informed about how environmental exposure may influence material behaviour.

Sub Goal 3: Protect Biological Data and Privacy While microbiome data may be used to generate initial material patterns, governance should ensure abstraction, anonymisation, and minimisation of biological data. Prevent misuse, re-identification, or unintended interpretation of microbiome information during research, development, or documentation.

GOAL 3: Design Collaboration and Material Interface This project treats biological systems not as tools, but as collaborators in material expression.

Sub Goal 1: Encourage Interpretive Ambiguity Frame material expression in a way that remains open and personal, avoiding fixed meanings or biological judgments. Patterns, colors, and textures should be experienced as aesthetic and reflective rather than explanatory, allowing wearers to form their own understanding over time.

Sub Goal 2: Fostering Long Term Embodied Relationships Encourage design approaches that support lasting engagement with the garment. The material should evolve gradually with the wearer and their environment, promoting attachment, care, and continuity rather than novelty or performance monitoring.

Sub Goal 3: Respecting Living Organisms and Collaborators Ensure that biological organisms involved in the design process are treated as collaborators rather than passive tools. This includes designing with respect for uncertainty, emergence, and biological behavior, and valuing learning and responsiveness over control.

GOAL 4: Diversity and Social Value

Sub Goal 1: Avoid Biological Hierarchies Design the system to celebrate microbial diversity and variation rather than framing certain biological compositions as superior, healthier, or more desirable.

Sub Goal 2: Inclusive and Contextual Design Account for diverse bodies, skin types, climates, cultures, and environmental contexts. Recognise that microbiomes vary widely and meaningfully across individuals and environments.

Sub Goal 3: Innovation and Long-Term Social and Cultural Value Promote designs that cultivate appreciation for inner biological diversity and interdependence with the environment, positioning fashion as a medium for ecological awareness and long-term reflection rather than critique or correction of the body.

GOVERNANCE ACTIONS AND EVALUATION

Governance Action 1: Abstraction and Use of Microbiome Data

Purpose: Currently, biological data, especially DNA, is often treated as a source for identity and diagnosis. This governance action proposes using microbiome data solely to reflect and generate material patterns for a “custom skin design,” rather than as a health monitor. The goal of the design is to enable personalisation of wearables to complement identity through one’s personal microflora without reinforcing medicalised interpretations of the body’s health states over time (Nuffield Council on Bioethics).

Design: Through a design lens, this would require that microbiome data is reduced to high-level non-identifying parameters and indicators, before translating material forms, textures and colours. Academic researchers and designers would document this abstraction process through an ethical review, and institutions or collaborators would approve projects only with clear boundaries between the user and their biological content being used to drive material changes and custom patterns.

Assumptions: This approach assumes that abstraction can preserve the richness of biological variation without revealing sensitive or identifying information. It also assumes users can connect with such a textile and material without it immediately reflecting some information related to the state of their health and microflora. There is also an assumption that the material will not be contaminated by other genetic material upon exposure, thereby triggering changes in its material codes.

Risk of Failure and Success: This governance action can fail if the abstraction is not clearly communicated to viewers, and if viewers view the material as a marker revealing biological truths subject only and specifically to their genetic skin material. If successful, the material will invite an understanding of one’s microbiome, and one will feel connected to the material through their identity reflected in their own personal ‘second skin’ with encoded patterns.

Governance Action 2: Consent and Awareness Over Time

Purpose: As this textile is designed to reflect, react and evolve with one’s microflora over time, consent needs to surpass time rather than a one-time agreement. This governance action aims to ensure that users are always informed and in control of their own genetic material as it interacts, changes, and reveals patterns of their skin over time (Nuffield Council on Bioethics).

Design: Users should be clearly informed about how the material of their genomic and microbial makeup is used and how it may change over time. They should also be informed of the factors related to those changes like triggers in the environment, temperature, ph, and even their diets. Users should understand consent as ongoing, with the opportunity for disengagement over time in relation to material evolution.

Assumption: This assumes that users are engaging with the material over time and are interested in transparency through evolution rather than at a first-time basis in a finished product or object. It also assumes the designer understands most possible changes, allowing them to clearly communicate material changes with users. It also assumes most users will reflect similar patterns without understanding all potential microbiome reflections in a material, and that changes to material patterns are limited by encoded engineered material.

Risk of Failure and Success The action’s failure is indicated if the user feels overwhelmed by their personal information and biological data and disengages with their custom skin. If successful, the material is always engaging and evolving, inspiring surprise, wonder, and a sense of a “custom” look, without the need for other satisfactory components, as in “fast fashion”.

Governance Action 3: Design for Long-Term Biological Relationship rather than one-time use / Rapid Consumption

Purpose: Fashion systems today value speed, novelty, and constant replacement, encouraging users to constantly change and seek satisfaction. This governance action proposes an alternative model in which biointeractive textiles are designed to evolve slowly with the wearer and form a long, healthy, symbiotic relationship, rather than providing short-term satisfaction. The purpose of this textile is to form a personalised system rooted in biological time, where changes are driven by the constant dialogue between the body’s microbiome and its environment.

Design: This requires the design to be bioengineered to reflect data from the microbiome first as a template for a custom DNA fingerprint, and then as a gradual system that transforms over use and time. This enables the user to grow a deeper relationship with the material over time. This reflects the design philosophy of patina, where ageing with the material becomes more valuable than a static material or object. Cultural institutions, as well as Art and Design communities and academic institutions, could support this design approach and change the language and cultural dialogue in how materials are approached. Evaluating success through longevity, attachment, and stewardship should be the leading criteria when designing responsibly, rather than focusing on scalability.

Assumptions: This assumes that wearers have the ability to care for slower forms of engagement over time rather than shift from one-time use excitement and satisfaction. It also assumes that the fashion system can function on individual personalised systems rather than consumable production cycles and that biological change over time can be experienced as meaningful rather than unpredictable and inconvenient.

Risks of Failure and Success: Failure could be seen where users could expect instant personalisation and are impatient with gradual changes over time. There is also the risk of market pressures reintroducing fast fashion logic and trying to accelerate production in biological processes that take time and are not scalable. If successful, the approach is culturally accepted and appreciated through a patina philosophy of design lens. The approach also challenges existing fashion paradigms and shifts into a niche that longs for custom-fitting products targeting individuals’ ecosystems rather than the masses.

POLICY GOALS RUBRIC

Rubric Backgroung Image Credit: Jonathan Williams and Paula Aguilera https://awomensthing.org/blog/neri-oxman-organic-design/#google_vignette

PRIORITIZED GOVERNANCE OPTIONS, TRADE-OFFS AND RECOMMENDATIONS

Drawing on the rubric, I would prioritise Option 3: Designing for a Long-Term Biological Relationship (Biological Time and Care), supported by Option 1: Abstraction First Use of Microbiome Data (Abstraction Data) and Option 2: Ongoing Consent and Lifecycle Transparency as essential safeguards (Ongoing Consent). Option 3 emerges as the strongest core strategy because it most directly supports the project’s fashion-specific and cultural goals: encouraging longevity over replacement, resisting fast-fashion consumption models, and framing the garment as a living system that gains meaning through time and use. By embedding biological time and co-evolution into the design, this option aligns with respect for living systems and positions fashion as a practice of care and stewardship rather than optimisation or novelty.

However, Option 3 alone would be insufficient without the protections offered by Options 1 and 2. Option 1 is critical in preventing health judgments, biological hierarchies, or genetic determinism by ensuring that microbiome data is abstracted before use and never interpreted diagnostically. Option 2 complements this by protecting user agency over time, recognising that consent must remain ongoing as the material evolves and as the relationship between wearer and garment deepens.

The primary trade-off in prioritising Option 3 is feasibility and scalability. Designing garments that evolve slowly and unpredictably may challenge existing fashion production models and limit immediate commercial adoption. There is also uncertainty around how users will respond to gradual biological change rather than instant personalisation. Nevertheless, these limitations are consistent with the project’s intent to change how we approach design as a living and growing system rather than a mass-market solution.

One key ethical concern is the risk of biological over-interpretation, particularly how easily biological data, especially DNA, can be read as defining identity, health, or value. Even when design intent is non-diagnostic, audiences may project meaning onto biological materials, raising concerns around determinism and surveillance.

Another concern involves time and consent. Working with living or evolving materials challenges traditional notions of informed consent, which are often treated as one-time agreements. This raises questions about how users remain informed and empowered as materials evolve over time.

Finally, the project highlights tensions between care and control in the design of living systems. Treating organisms as collaborators rather than tools requires accepting uncertainty and resisting extractive design instincts, an ethical shift that challenges dominant engineering and fashion paradigms as well as approaches to design.

To address these concerns, the governance actions proposed, particularly abstraction-first data use and ongoing consent, serve as mechanisms to limit overreach, preserve user agency, and maintain ethical boundaries around interpretation. Additionally, clearly framing bio-interactive textiles as cultural and expressive systems rather than medical or wellness technologies helps protect both users and designers from unintended misuse or misclassification.

This project aims to redefine textile paradigms by reframing how we understand and engage with what we wear. By connecting wearers to their own living micro-universe and genetic codes, the garment invites a deeper appreciation of both who they are and how they are evolving. Through daily use, the material develops its own patina, not through wear alone but through biological change, encouraging an ongoing relationship with the textile. In doing so, the project fosters care and respect for the body as a dynamic system, and for the microbial worlds that support our skin’s rich ecosystem and for the environments that continuously shape it.

REFERENCES:

Atallah, C., El Abiad, A., El Abiad, M., Nakad, M. and Assaf, J.C. (2025) ‘Bioengineered Skin Microbiome: The Next Frontier in Personalized Cosmetics’, Cosmetics, 12(5), p. 205. doi: 10.3390/cosmetics12050205.

Marvin, J. (2016) Between the Chisel and the Gene: Neri Oxman’s Organic Design. A Women’s Thing. Available at: https://awomensthing.org/blog/neri-oxman-organic-design/

National Institutes of Health (NIH) (2026) Human Microbiome Project. Available at: https://hmpdacc.org

Nuffield Council on Bioethics (2026) Nuffield Council on Bioethics. Available at: https://www.nuffieldbioethics.org

Week 2 HW: DNA Read Write & Edit

Part 1: Benchling & In-silico Gel Art

https://benchling.com/s/seq-B1mFk0Oh2ZF9coVeqBMH?m=slm-ySVijsqZThHxK3Qq6kwq

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

I am interested in the protein Reflectin. This protein is common in Cephalopods and is responsible for their structural colour as their skin changes with the environment. I am interested in the potential applications of Reflectin in smart materials/textiles. Reflectins’ optical properties can be reversibly engineered to change colour under different conditions. There is one study in which Reflecin was used in this way in a thin film substrate, and responsive to hydration or dehydration of the material.

Protein Sequence:

tr|Q6WDN6|Q6WDN6_EUPSC Reflectin 3a OS=Euprymna scolopes OX=6613 PE=4 SV=1 MNRYMNRFRNFYGNMCRNRNRGMMEPMSRMTMDFQGRYMDSQGRMVDPRYYDYYGRYNDY DRYYGRSMFNYGWMMDGDRYNRYNRWMDYPERYMDMSGYQMDMYGRWMDMQGRHCNPYSQ WMMYNYNRHGYYPNYSYGRHMFYPERWMDMSNYSMDMYGRYMDRWGRYCNPFYHYYNHWN RSGNNPGYYSYYYMYYPERYFDMSNWQMDMQGRWMDMQGRYCSPYWYNWYGRQMYYPYQN YYWYGRWDYPGMDYSNWQMDMQGRWMDMQGRYMDPWWMNDSYYNNYYN

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

reverse translation of tr|Q6WDN6|Q6WDN6_EUPSC Reflectin 3a OS=Euprymna scolopes OX=6613 PE=4 SV=1 to a 864 base sequence of most likely codons. atgaaccgctatatgaaccgctttcgcaacttttatggcaacatgtgccgcaaccgcaac cgcggcatgatggaaccgatgagccgcatgaccatggattttcagggccgctatatggat agccagggccgcatggtggatccgcgctattatgattattatggccgctataacgattat gatcgctattatggccgcagcatgtttaactatggctggatgatggatggcgatcgctat aaccgctataaccgctggatggattatccggaacgctatatggatatgagcggctatcag atggatatgtatggccgctggatggatatgcagggccgccattgcaacccgtatagccag tggatgatgtataactataaccgccatggctattatccgaactatagctatggccgccat atgttttatccggaacgctggatggatatgagcaactatagcatggatatgtatggccgc tatatggatcgctggggccgctattgcaacccgttttatcattattataaccattggaac cgcagcggcaacaacccgggctattatagctattattatatgtattatccggaacgctat tttgatatgagcaactggcagatggatatgcagggccgctggatggatatgcagggccgc tattgcagcccgtattggtataactggtatggccgccagatgtattatccgtatcagaac tattattggtatggccgctgggattatccgggcatggattatagcaactggcagatggat atgcagggccgctggatggatatgcagggccgctatatggatccgtggtggatgaacgat agctattataacaactattataac

3.3. Codon optimization.

Optimized Codon Sequence

Using Yeast (Pichia Pastoris)

While E-Coli is a great organism to use for simple codon optimization, in this case with the codon optimization for Reflectin 3a Protein, Yeast ‘Pichia Pastoris’, is a better choice for a more complex fold and protein like Reflectin a more sufisticated and complex eukaryotic protein.

1 ATGAATAGAT ACATGAATAG GTTTAGAAAC TTCTACGGAA ACATGTGTAG GAACAGAAAT AGGGGAATGA 71 TGGAACCTAT GTCCAGGATG ACAATGGACT TCCAAGGAAG GTATATGGAC TCTCAAGGTA GGATGGTTGA 141 TCCTAGGTAC TACGACTACT ACGGTCGTTA TAACGACTAC GACAGATACT ACGGTAGATC TATGTTCAAT 211 TACGGTTGGA TGATGGATGG TGACAGGTAT AATAGATACA ACCGTTGGAT GGATTACCCC GAAAGGTACA 281 TGGATATGAG TGGATATCAA ATGGATATGT ATGGCAGATG GATGGATATG CAAGGAAGAC ATTGCAACCC 351 ATACTCACAA TGGATGATGT ACAATTATAA CAGGCACGGT TATTATCCTA ACTATTCCTA CGGCCGACAT 421 ATGTTCTACC CTGAACGTTG GATGGACATG TCTAACTATT CAATGGACAT GTATGGAAGA TACATGGATA 491 GGTGGGGAAG GTACTGCAAC CCATTTTACC ACTACTATAA CCATTGGAAC AGAAGTGGAA ATAATCCTGG 561 CTACTACTCC TACTACTATA TGTACTATCC CGAGAGATAC TTCGACATGT CCAACTGGCA GATGGACATG 631 CAAGGAAGAT GGATGGATAT GCAAGGAAGA TATTGTTCTC CTTACTGGTA TAACTGGTAT GGTAGACAGA 701 TGTACTATCC ATATCAGAAT TATTATTGGT ACGGAAGGTG GGATTATCCT GGAATGGATT ACTCCAATTG 771 GCAGATGGAC ATGCAAGGTA GATGGATGGA CATGCAAGGC AGATACATGG ATCCATGGTG GATGAACGAC 841 TCCTACTACA ATAACTACTA TAAT

3.4. You have a sequence! Now what?

Reflectin, as a protein, has many potential applications, especially in textiles. Once the yeast has harvested the reflectin protein with optimised codons for yeast Pichia Pastoris, the reflectin protein can be purified and used as a structural colour material in textiles, with multiple different gradients. Relfectin could also be engineered to be expressed in cells with biological signals. In the same way that reflectin is used in cephalopods to express a change in environment in response to stimuli, Reflectin can be engineered into a material that responds to its environment. Reflectin also has electrical and dialectic properties which could be used for bioelectronic systems and technologies. There is increasing research on the future of bioelectronics, including deriving electrical signals from biological organisms and engineered systems in which they are embedded and used to power systems. In the case of Reflectin, it can be incorporated into a bioconductive system, for example, EMG, and respond to one’s own muscular electrical pulses, changing depending on one’s skin conductivity,(Cai.T). Skin conductivity can also reflect one’s neurological system, thereby revealing one’s internal worlds and expressing emotions such as excitement and adrenaline.

Part 4: Prepare a Twist DNA Synthesis Order

4.2. Build Your DNA Insert Sequence

Expression cassette https://benchling.com/s/seq-gDhXeHq7OCFYtyBIH9bE?m=slm-7Ec7akbFEDQuYeDYt6eK

4.3. On Twist, Select The “Genes” Option

Part 5: DNA Read/Write/Edit

(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 would like to sequence the ‘16S rRNA gene’. This DNA is found in the skin microbiome and serves as a diversity metric for the bacteria in our skin’s gut and more! As we are all made of a diverse ecosystem of bacteria, I would love to further extract one’s metrics from this DNA, which serves as a ‘bacterial barcode’; it is also a cheap and efficient DNA to work with. Additionally, instead of sequencing a single DNA sample, I can sequence all microbial DNA in a single sample. This makes 16S a very efficient DNA. With its diverse micropalette, I would love to explore its potential for applications in materials that reflect one’s unique microflora.

(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?

To perform the DNA edit of the 16S rRNA gene, I would choose the Illumina next-generation sequencing technology as 16S regions are short (250-500bp), and Illumina gives high accuracy, as well as allowing a multiplexity of samples. For more comprehensive functional analysis through whole metagenomic sequencing sampling, I would pick the Oxford Nanopore for long sequencing for long reads to improve genome assembly and detect structural variations. Together, these technologies can provide a functional analysis of the DNA and its microbial species.

Also answer the following questions:

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

Illumina sequencing is a second-generation ’next-generation’ sequencing technology. It is considered second-generation due to a few factors. Firstly, it performs massively parallel sequencing, where DNA fragments are sequenced simultaneously; it also relies on sequencing by synthesis of clonal amplification. This is unlike technologies like Sanger, which sequence one fragment at a time, and require PCR amplification.

The preparation steps begin with collecting microbial DNA from skin swabs. Next, total microbial DNA is extracted through cell lysis and purification. The 16S rRNA hypervariable regions are then amplified using PCR with region-specific primers. In a subsequent indexing PCR step, Illumina adapters and sample-specific barcodes are added to both ends of the amplicons to enable multiplexing and flow cell binding. The amplified libraries are purified to remove primer dimers and unwanted fragments, quantified, normalised, and pooled. The final product is an adapter-ligated DNA library ready for cluster generation and sequencing on an Illumina platform.

Illumina decodes DNA through ‘sequencing by synthesis’. Flourecently labeled nucelotideswith reversible terminators are incorporated one base at a time. Fluorescent imaging then detects each nucleotide. The software then converts the visible fluorescent signals into base calls (A, C, T, G). The output is millions of sequence reads stored as FASTQ files, including all nucleotide sequences and quality scores for each base.

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?

Based on the DNA sequencing of 16S, and identifying the microuniverse, microflora and diverse rich ecosystems of one’s skin, I would edit specific pigment biosynthesis in microbial genes to produce select pigments, creating chromatic responses in a textile. I would insert promoters responsive to environmental factors such as pH, humidity, or metabolite concentration. The engineered microbes could then produce pigment in dynamic gradiations according to the wearer and their environment. This design interface approach leverages gene editing as a translational medium for expressing microworlds as a visual living and smart material.

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

To perform this gene edit, the use of a CRISPR-Cas9 system would be needed for pigment biosynthesis. Crispr Cas9 can offer precise base pair editing of promoter regions, tuning expression without breaking the double strand. Editing machinery into bacterial cells could be achieved via electroporation or conjugative plasmids. Inducing pigment production responsive to the environment, synthetic gene circuits would be engineered through inducible promoters or quorum-sensing systems. PCR sequences would also need to be validated edited strains before integrating into a bioresponsive textile system.

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

Cas9 is an RNA-guided endonuclease which induces a double-strand break at a target DNA sequence chosen by the complementary guide RNA and recircularized PM site. The Cas9 enzyme then cuts the DNA in the target site. The cell then repairs the cut, and DNA sequences can be changed during the repair by disrupting the gene or inserting a new sequence with a provided repair template. For the editting process I would first need to design an RNA reciprocale with the target gene and ensure a PAM site is close to the target. The input would contain a plasmid including the CAS9, guide RNA, bacterial cells and the repair template. The process would first include the plasmid construction, introducing the plasmid into cells, CRISPR cutting the DNA, and lastly selecting the successful edits of the cell.

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

One limitation of CRISPR-Cas9 editing tools is the potential for off-target effects, in which the guide RNA partially binds to unintended genomic regions, leading to unintended DNA cuts. Also, as this technology requires a PAM site near the target site, it limits the locations that can be edited. The cell’s DNA repair mechanisms can also reduce efficiency, as homology-directed repair may often be unpredictable and can require screening of multiple clones. Efficiency can also be reduced by the delivery of CRISPR components into bacterial cells, especially in non-model organisms. Lastly, viability may be reduced by continuous double-strand breakage, which can be toxic to cells and thus reduce viability.

RESOURCES:

Cai, T., Han, K., Yang, P., Zhu, Z., Jiang, M., Huang, Y., Xie, C. (2019) Reconstruction of dynamic and reversible color change using reflectin protein. Scientific Reports, 9, 5201. Available at: https://www.nature.com/articles/s41598-019-41638-8

Week 3 HW: Lab Automation

Post-Lab Questions

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

PUBLICATION:

The publication Semiautomated Production of Cell-Free Biosensors, published by the American Chemical Society (ACS Publications), explores the assembly of cell-free biosensors through liquid handling robotics versus manual methods commonly used in lab-scale development. The process is a combination where, “both manual and semiautomated reaction assembly approaches using the Opentrons OT-2 liquid handling platform on two different cell-free gene expression assay systems that constitutively produce colourimetric (LacZ) or fluorescent (GFP) signals,(Brown).” The deigned protocols demonstrate that they perform close to expected detection outcomes, in a more controlled environment, (Brown).

Copyright © 2025 American Chemical Society

IDEA:

In my final project, I intend to use automated fabrication tools to create 3d printed templates, moulds, and matrices within the fabric, as well automating the liquid handling in the textile. Through automation, I will construct a textile embedded with patterned cavities designed to host distinct bacterial-sensing environments. The fabric’s automated structure serves as the fixed framework, while the bacteria and their metabolic activity constitute the variable component. As microbial signals interact with each cavity, dynamic changes in colour and pattern emerge, allowing the fabric to visually reflect microbial activity and ecological variation across its surface.

To do this, the following steps are needed.

1. Opentron OT-2 liquid handling and 3d Printed textile: First, I need to design the 3D mould for the hydrogel fabric textile, which will consist of a cavity matrix that will host the cell-free biosensors. I also need to 3d print a holder to lay the textile flat and dispense to OT-2 coordinates.

2. Bioprinting hydrogel: Next, I need to print the hydrogel containing the pattern cavities designed and corresponding to the OT-2 dispense coordinates. This can be done by bioprinting or mould casting the hydrogel into shape/texture.

3. Opentrol OT-2 despensing: The Opentron OT-2 will despense the DNA mixture and CFE into the bioprinted hydrogel cavities and coordinates. This will ensure accurate volume, distribution, and reproducibility across various textile matrices.

4. Sealing of textile hydrogel: The textile hydrogel will be sealed with a semipermeable layer that allows skin metabolites to permeate into the cavities and activate cell-free biosensors to reveal patterns.

PROJECT PROPOSALS: https://docs.google.com/presentation/d/19T22uOQz9aTvnzh426tri4xrg-ZoG1ksy6I5bswGHaw/edit?usp=sharing

RESOURCES:

Brown, D.M., Phillips, D.A., Garcia, D.C., Arce, A., Lucci, T.J., Davies, J.P., Mangini, J.T., Rhea, K.A., Bernhards, C.B., Biondo, J.R., Blum, S.M., Cole, S.D., Lee, J.A., McDonald, N.D., Wang, B., Perdue, D.L., Bower, X.S., Thavarajah, W., Karim, A.S., Lux, M.W., Jewett, M.C., Miklos, A.E. & Lucks, J.B., 2025. Semiautomated production of cell-free biosensors. ACS Synthetic Biology, 14(3), pp.979–986. doi:10.1021/acssynbio.4c00703.

Chowdhury, M.-U.-S., Roy, S., Kumar, A., Kakadiya, D., Deshpande, G. G., Aryal, K. P., Leung, H. & Pandey, R., 2025. Development of a conductive fabric-based wearable patch for multiplexed measurement of sweat glucose and sweat secretion. Preprint. Research Square.. https://www.researchgate.net/publication/393943187_Development_of_a_Conductive_Fabric-based_Wearable_Patch_for_Multiplexed_Measurement_of_Sweat_Glucose_and_Sweat_secretion

Horland, R., Lindner, G., Wagner, I., Atac, B., Hoffmann, S., Gruchow, M., Sonntag, F., Klotzbach, U., Lauster, R. & Marx, U., 2011. Human hair follicle equivalents in vitro for transplantation and chip-based substance testing. BMC Proceedings, 5(Suppl 8), p.O7.https://pmc.ncbi.nlm.nih.gov/articles/PMC3284944/?utm_

SOL – Seed Of Life, 2018. 3D Printed Wearables Bio-Engineered with Bacteria That Can Embed Living Matter, 8 June 2018. Psychedelic Clothing & Visionary Art blog. https://www.psytshirt.com/blog/psychedelic-fashion-clothing-trippy-t-shirt-seed-of-life-3D-Printed-Wearables.html?srsltid=AfmBOoptLpo7rSj0967utQ-LieopClYKuq6HHlT0tg63bBcr-ivp7RZO

Week 1 Lab: Pipetting

Individual Final Project

Group Final Project