Analog photography has been experiencing a growing revival and with it a growing ecological concern, specially regarding the impacts of its “magical” component — silver halides. Much of the movement of trying to address the environmental impact of analogue film has fallen on individual artists and researchers, by trying to mitigate the consequences of silver. However, despite the efforts of exploring plant-based developers, and darkroom procedures to prevent damaging disposal of silver contaminated solutions, (extremely toxic for the environment affecting primarily microbial life) we are still left with the need to use this toxic metal in lack of any other option for analog camera photography.

Based on chlorophyll’s photosensitive effectiveness, my research is focused on exploring this molecule as an alternative substance to silver. As of now, I have reached the conclusion that there is a potential in this molecule due to the process of degradation that occurs when chlorophyll is exposed to light outside a living cell — it can demetallate into a porphyrin-type structure that is able to chelate iron, therefore, creating a negative image formed by iron complexes. This hypothesis uses chlorophyll as the photosensitizer and iron as the density builder in order to obtain an image. By developing the image with iron and creating porphyrin-iron type complexes it’s possible to confer a permanent image formation — archival quality to be tested.

For this effect I would like to explore the possibility of engenineering bacteria that could produce a modified version of chlorophyll (that could be organized into supramolecular structures) for optimal photographic application, or an adapted version light-harvesting chlorophyll proteins (LHCPs). The use of bacteria for this effect would ensure a renewable efficient way of producing a photographic emulsion at industrial level.

In a more speculative note, there has also been a growing interest in the experimental photography community to use SCOBY membranes as photographic printing support. It would be interesting if the bacteria could be designed to form a chlorophyll layer at the surface of the cellulose membrane in order to grow photographic “paper”.

Observed: Chlorophyll photodegradation, porphyrin demetallation, iron chelation

Speculative: supramolecular organization of chlorophyll, engineered LHCPs for photographic purposes, SCOBY-grown photographic paper

This intersection between biotechnology and the foundation of an artistic medium can incentivize the much-needed discussion around the role of art when confronted with technological advances and the revision of artistic practices. Specially in the context of ecological artistic practices, there is an interesting space to explore the limits of what is considered ethical in order to make the most out of other-than-human interactions and the creation of symbiotic links through biotechnology.

Although this project is primarily focused on a material design/engineering point of view and the possible development of a new photographic process it does pose ethical questions both at practical and conceptual levels. Specially if entertaining the idea of a photographic SCOBY, that could be passed from one enthusiast to another like it happens today with kombucha cultures. Still, even if we just contemplate the possibility of a genetic modification derived chlorophyll film, that would no longer contain living cells, there could be some implications at conceptual levels regarding people that are developing ecological practices. Taking this into account, some governance/policy goals that could make this project come to life in a safe and ethical manner are:

Transparency regarding the modifications

Have open documentation of genetic modifications in the cells used and processes of production of the film, to allow for an informed ethical evaluation by the users.

The same would apply to a SCOBY plus clearly stating what living cells would that culture contain.

Esurance of biosafety

Utilization of bacteria that present low biosafety hazard risk, both for human handling and eventual environmental release.

Create clear protocols of disposal and deactivation of the cultures.

Understand the impact of a modified culture that could be grown and passed from one person to another in an amateur context

Use of a “kill switch” – nutrient without which the SCOBY culture couldn’t survive

Environmental sustainability

Understanding the life cycle of the engineered material and create clear protocols for sustainable use

Design the materials that compose the film to ensure biodegradability, like substituting the gelatine used in current films for algae derived gels and using bioplastic as film base.

Avoid “greenwashing” through a narrative of sustainability without being sure of the extent of the possible impacts

Pedagogy and discussion

Generate open discussions about ethical use of synthetic biology and offer workshops on the use of this technology

Use as teaching tool to contribute to a more distributed knowledge about biotechnology and how it can be used for creation and evolution. The SCOBY could be a great opportunity to demystify synthetic biology.

Preventing misuse or misinterpretation

While trying to democratize the knowledge about synthetic biology the take measures to prevent the public notion of biological = harmless

Preventing unregulated bio-modification

Thinking about these subjects made me understand the difficult role of defining where should be the limit in making synthetic biology more accessible to the public and made me eager to start dialogues around the role of the arts in play in this. I still think it is important to show how humans can relate with other species at different levels and through symbiosis (whatever form it takes) evolve

Action 1(most plausible): Disclosure standard for bio-engineered materials

Actors: Academic and Scientific researchers, Art institutions, Funding institutions

Purpose: Regarding the case of the chlorophyll film which could be produced through GM bacteria, but the final product wouldn’t contain any viable » cells. This, by Portuguese and EU law wouldn’t have the need of any labeling regarding its origin of production. As a result, users and audiences are typically unaware of the biological engineering involved in the production process. Proposal: Introduce a voluntary disclosure standard for this type of product outside the food and feed context that are derived from GMO production processes but are themselves non-living, clarifying their origin.

Design:

• Develop a standardized disclosure label or documentation stating: That the material was produced using genetically modified microorganisms under contained conditions The procedure of production: which type of organism was used and in which material it contributed to That the final product contains no living organisms or viable genetic material
• Adoption driven by: Research institutions Art schools, Museums and Galleries Funding agencies — transparency statements

Assumptions:

• It’s expected that transparency of production methods increases trust rather than fear
• Artists, researchers and institutions are willing to adopt ethical commitments
• The public will be receptive to the difference between process-based and product-based genetic modification when explained clearly

Risks of Failure & Sucess: Failure:

• Low adoption due to lack of incentives
• Misinterpretation of disclosure as associated risk Success:
• Voluntary disclosure could become an informal requirement
• May unintentionally reinforce the idea that GMO-derived products are Inherently suspect

Action 2 (still plausible): Offering demonstrations in contained conditions

Actors: Researchers; Selected Laboratories (could be biolabs); Experimental Photography Organizations; Public — artists, enthusiasts

Purpose: Regarding both the chlorophyll film and the SCOBY.

Proposal: Through the reach of international photography organizations arrange in collaboration with biolabs demonstrations of the production of both the film and the live photographic SCOBY in contained conditions.

Design:

• According to biosafety levels of both the needed GMOs and the laboratories it should be possible to realize demonstrations of the chlorophyll film production using modified bacteria and the growing of photographic SCOBY membranes since these wouldn’t leave biosafety areas.
• These demonstrations could include the following: The protocols for extracting the modified chlorophyll from the bacteria and turning it into photographic emulsion Developing and processing chlorophyll film Overview the safe and sustainable disposal of the film and chemicals used The protocols used to grow the photographic SCOBY membrane such as feeding, processing the grown membrane, print an image on it and develop it.
• This would be a great opportunity to be able to understand the opinion of the artistic community regarding the use of synthetic biology. >And if it seems justifiable for this end.

Assumptions:

• By sharing the production protocols of a new analog photography technology artists might be more interested to build upon it and feel more confident about biotechnology
• The interaction with a living GM SCOBY would largely contribute for the demystification of synthetic biology
• The public interested in both traditional analog photography and experimental photography would be available to understand more about a new and ecological way of using film

Risks of Failure & “Success” Failure:

• Lack of adherence due to preconceived ideas about GMOs and ethical collision against ecological practices Success:
• Increase of concerns about GMOs due to the demonstration being restricted to biosafe infrastructures

Action 3 (least plausible): Framework for release and sharing of GM SCOBY

Actors: Portuguese and EU regulatory bodies; Research centers; Community Labs

Purpose: Under Portuguese and EU regulation, the deliberate release of genetically modified organisms into the environment — including sharing living cultures outside contained laboratory conditions — is heavily restricted and prohibited without formal authorization. Informal circulation of living GMOs through artistic or DIY communities is not legally accommodated. Proposal: Establish a formal regulatory framework that would allow, under strict conditions, the deliberate release and downstream sharing of that genetically modified SCOBY, used for artistic or photographic purposes.

Design:

• Develop a dedicated authorization pathway under existing GMO delierate release legislations adapted for non-agricultural, non-food, artistic uses
• Requirements would include: Environmental risk acessment Proof of containent or ecological self-limitation Monitoring and reporting obligations Clear disposal protocols
• Oversighted by the Portuguese regulator (APA), possibly coordinated at the EU level
• Participation could imply institutional support from an university/research center, community biolabs for approval prior to sharing or release

Assumptions:

• The environmental risks of a modified SCOBY can be sufficiently predicted and controlled
• Regulators would be willing to differenciate cultural/artistic uses from agricultural and and commercial uses
• That downstream user would comply with handling, propagation and disposal protocols
• That a legal framework would reduce informal or illegal dissemination

Risks of Failure & “Success” Failure:

• High admnistrative and financial burden could make the framework unusable
• Difficulty in ensuring compliance once organisms start circulating
• Public opposition to deliberate release of GMO undermines feasibility Success:
• Normalising the release of GMO for artistic porpuses could endanger biosafety norms
• Authorized release could be interpreted as being biologically harmless and contribute to reckless use
• Aproval could legitimize risky practices under the context of art

Disclosure standard for bio-engineered materials

This action would receive the most priority since having a disclosure that allows the consumer to make an informed ethical decision about the technology offered is essential and creates an opportunity to broaden the perception of the range of synthetic biology use.

Offering demonstrations in contained conditions

I consider this action the most interesting to accomplish the main two objectives of increasing proximity and dissemination of biotechnology in the arts and incentivize research on the topic of this project. However, it would imply more difficulties due to the need for living GM cultures, that not being authorized to leave biosafe areas, would need to be reproduced in every lab the demonstrations took place.

First of all I started by making a digest with a single enzyme at a time.

Then tried to color code the result of every enzyme and superimpose them on top of each other so to create a “grid” were I would make my design. I soon understood it would be way too confusing, plus, that the result of using a combination of enzymes doesn’t necessarily correspond to the superimposition of the lines created by each enzyme separately.

Then I started using Ronan’s website to iterate on combinations of enzymes + using some unconventional techniques.

The protein I’d like to work with is the prochlorophyte chlorophyll-binding (Pcb) protein which is the light-harvesting protein (LHP) in prokaryotes that uses only chlorophyll as their photosensitive pigment. A modified version of this protein could be used to efficiently absorb light causing degradation of chlorophyll a, b and d molecules into porphyrin-type derivatives — that can be used to bind iron and create photographic images in a cell-free system.

MGMQTYGNPDVEYGWWAGNSRLAGFSGKWLAAHVAQAALIVFWAGAICLFEVARYTADVPLGEQNLILIPHMASLGLGIGEGGQIVDTFPYFAVGVVHLVSSAVIGAGGLYHSLRGPAILKEGPARAPKFDFDWGDGKRLGFILGHHLILLGLGALFLVLWAVFFGIYDPVIGEVRTVTSPTLNPFTIFGYQTHFVETNTLEDLIGGHVYVAIIEISGGLWHIFCPPFKWAQRLIIYSGEGLLAYALGGLAIMGFTAAVYCAFNTLAYPVEFYGPPLDFRFSFAPYFIDTADLPSGQYTARAWLCNVHFFLAFFVLQGHLWHALRTLGFDFKRIPAALGSLSEDVVDAKA

(from NCBI chlorophyll a/b binding protein [Prochloron didemni])

ATGGGCATGCAGACCTATGGCAACCCGGATGTGGAATATGGCTGGTGGGCGGGCAACAGCCGCCTGGCGGGCTTTAGCGGCAAATGGCTGGCGGCGCATGTGGCGCAGGCGGCGCTGATTGTGTTTTGGGCGGGCGCGATTTGCCTGTTTGAAGTGGCGCGCTATACCGCGGATGTGCCGCTGGGCGAACAGAACCTGATTCTGATTCCGCATATGGCGAGCCTGGGCCTGGGCATTGGCGAAGGCGGCCAGATTGTGGATACCTTTCCGTATTTTGCGGTGGGCGTGGTGCATCTGGTGAGCAGCGCGGTGATTGGCGCGGGCGGCCTGTATCATAGCCTGCGCGGCCCGGCGATTCTGAAAGAAGGCCCGGCGCGCGCGCCGAAATTTGATTTTGATTGGGGCGATGGCAAACGCCTGGGCTTTATTCTGGGCCATCATCTGATTCTGCTGGGCCTGGGCGCGCTGTTTCTGGTGCTGTGGGCGGTGTTTTTTGGCATTTATGATCCGGTGATTGGCGAAGTGCGCACCGTGACCAGCCCGACCCTGAACCCGTTTACCATTTTTGGCTATCAGACCCATTTTGTGGAAACCAACACCCTGGAAGATCTGATTGGCGGCCATGTGTATGTGGCGATTATTGAAATTAGCGGCGGCCTGTGGCATATTTTTTGCCCGCCGTTTAAATGGGCGCAGCGCCTGATTATTTATAGCGGCGAAGGCCTGCTGGCGTATGCGCTGGGCGGCCTGGCGATTATGGGCTTTACCGCGGCGGTGTATTGCGCGTTTAACACCCTGGCGTATCCGGTGGAATTTTATGGCCCGCCGCTGGATTTTCGCTTTAGCTTTGCGCCGTATTTTATTGATACCGCGGATCTGCCGAGCGGCCAGTATACCGCGCGCGCGTGGCTGTGCAACGTGCATTTTTTTCTGGCGTTTTTTGTGCTGCAGGGCCATCTGTGGCATGCGCTGCGCACCCTGGGCTTTGATTTTAAACGCATTCCGGCGGCGCTGGGCAGCCTGAGCGAAGATGTGGTGGATGCGAAAGCGTAA

(converted using bioinformatics.org)

Codon optimization is an important process due to different organisms having and producing different amino acids in different proportions. So, if a gene codes for a rare amino acid, it might slow the translation process and therefore the folding of the protein and might even render the protein non-functional. In this case, the gene should be optimized for e. coli which is probably the best choice since the primary objective is to express a protein that is going to be used in a cell-free system and it is the simplest organism to work with. For this codon optimization I avoided Type IIS enzyme recognition sites for BsaI, BsmBI, and BbsI — these are some enzymes that are useful for ligation with plasmid backone.

ATGGGGATGCAAACGTACGGAAATCCTGACGTAGAGTACGGTTGGTGGGCTGGAAATTCAAGATTAGCTGGATTCTCTGGTAAGTGGCTTGCAGCTCACGTAGCACAAGCCGCACTTATAGTTTTCTGGGCAGGTGCAATATGTTTATTCGAGGTCGCCCGTTACACAGCTGACGTCCCTTTAGGTGAGCAAAATCTTATCTTGATCCCACACATGGCTTCCTTAGGTCTTGGTATAGGAGAGGGTGGTCAAATCGTTGACACATTCCCATACTTCGCTGTTGGTGTCGTACACCTTGTTTCCTCGGCCGTCATCGGGGCAGGTGGTTTGTACCACTCTTTACGAGGTCCCGCCATATTAAAGGAAGGACCCGCACGTGCTCCAAAGTTCGACTTCGACTGGGGCGACGGTAAGCGGTTAGGATTCATCTTAGGTCACCACTTGATACTCTTAGGGTTAGGGGCCCTTTTCCTTGTACTTTGGGCAGTCTTCTTCGGTATATACGACCCTGTTATAGGGGAAGTAAGAACGGTTACATCCCCTACATTGAATCCATTCACAATATTCGGTTACCAAACTCACTTCGTAGAGACTAATACGCTTGAGGACTTAATCGGTGGTCACGTTTACGTCGCCATCATCGAGATCTCCGGCGGGTTGTGGCACATCTTCTGTCCCCCATTCAAGTGGGCACAACGATTGATCATATACTCAGGTGAGGGGTTGCTTGCCTACGCATTGGGTGGTCTCGCTATAATGGGTTTCACTGCCGCAGTCTACTGTGCCTTCAATACGCTTGCCTACCCTGTAGAGTTCTACGGTCCACCTTTAGACTTCCGTTTCTCATTCGCACCATACTTCATCGACACAGCCGACTTGCCGTCCGGGCAATACACAGCCCGAGCCTGGTTGTGTAATGTTCACTTCTTCTTAGCTTTCTTCGTATTGCAAGGGCACCTTTGGCACGCATTACGTACGCTTGGTTTCGACTTCAAGCGTATCCCCGCAGCATTAGGTTCCCTCTCTGAGGACGTTGTTGACGCCAAAGCGTAA

(made with Codon Optimization Tool | Twist Bioscience)

This gene sequence could be synthesized through chemical synthesis on silicon chips and assembled into a vector backbone— bacterial plasmid— then, put into e. coli. This allows for the use of the bacteria’s own cellular machinery to first transcribe this DNA sequence into mRNA (which would be identical to this coding DNA strand, except for “U”), and finally the bacteria’s ribosomes would translate the resulting mRNA into the final amino acid sequence. This AA sequence would be the pcbA protein in its apoprotein form— since e. coli lacks the machinery to produce chlorophyll molecules that play as a co-factor in the folding of this protein— which would be later combined with chlorophyll extract to render it functional.

1. Describe how a single gene codes for multiple proteins at the transcriptional level.

In biological systems, a single gene can code for multiple proteins at the transcriptional level through the process of alternative splicing (in eukaryotes)— a process where different combinations of exons from the same pre-mRNA molecule are joined together. This process happens inside the nucleus during the processing of pre-mRNA, leading to the synthesis of multiple protein isoforms, which are related forms of the same protein, but with different structural or functional properties. Another process that allows for a single gene to code for multiple proteins (both in eukaryotes and prokaryotes) is the action of alternative promoter genes that create different initiation sites, affecting which exons are included in transcription.

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

The DNA I’m interested in sequencing and further understanding is cyanobacteria’s genes for the production of Chlorophyll LHP (Light-Harvesting proteins) which is the nature’s way of organizing chlorophyll molecules in order to get the most light absorption out of them. This biological way of organizing light sensitive pigments could be the answer to a new generation of analog photography media and can also be used to engineer more efficient solar cells to produce energy.

For this type of application, the most adequate sequencing technology would probably be Sanger sequencing using a device like Sanger-ABI, a 1st generation technology that has been around since 1977 but would be more than enough for things like reading single protein coding sequences. This would be a small-scale project needing only to analyze relatively small nucleotide sequences, it wouldn’t demand the comparison of more complex genes like comparing/analyzing whole genomes. For this method I would make a DNA extraction from cyanobacteria cells and purify it, followed by designing primers specific for the sequence I want to analyze and amplify it through PCR, then remove excess primers and dNTPs. Next step would be to perform a cycle sequencing reaction “chain terminator PCR” using single primers, DNA polymerases, dNTPs and fluorescently labeled ddNTPs— these fluorescent ddNTPs act as chain terminators, stopping synthesis randomly at every possible length to create labeled fragments. Clean up residual dye labeled ddNTPs to prevent noise during read and submit these tagged fragments to Sanger-ABI capillary electrophoresis, which separates the fragments by length and then makes the read by exciting the label of each fragment and detecting the color emitted— then using software to translate the fluorescence signals into a chromatogram, revealing the sequence of the DNA sample.

I would need to synthesize a bacterial plasmid (for e. coli) with the insert for the Chlorophyll LHP gene, in this case the PcbA protein. The application for this would be to develop a way to keep these proteins functioning in a cell free system in order to create a novel biomaterial that would serve as photographic emulsion for analog film.

Probably the most affordable and effective way to synthesize this kind of DNA would be the through clonal gene chip-based chemical synthesis and assembly of the DNA sequence into a plasmid vector through Golden Gate Assembly using type IIS restriction enzymes and T4 DNA ligase. Due to the small-scale nature of this project and standard difficulty of synthesizing this kind of DNA, I don’t think there would be significant limitations speed, accuracy and scalability wise.

For the objective of this project the DNA that would be interesting to edit would range from the genes coding for the chlorophyll synthesis pathway— in order to develop a modified version of chlorophyl that would be more optimized for photographic purposes— and the sequences coding for the LHP if there is the need to modify the natural occurring proteins, either by decreasing the protection these proteins confer against chlorophyll degradation or by potentially improving on their ability to maximize chlorophyll’s light absorption qualities.

CRISPR-based genome editing would probably be the best choice for this purpose since Chlorophyll biosynthesis involves multiple genes, often with regulatory fine-tuning rather than simple on/off behavior. CRISPR systems allow gene-specific, locus-specific edits, making them well suited for altering enzyme functionality in the chlorophyll synthesis pathway; modifying regulatory regions that affect pigment ratios (e.g. chlorophyll a, b or d) and engineering specific amino-acid changes in LHPs. CRISPR edits DNA by using an RNA guide to bring a DNA-cutting enzyme (nuclease) to a specific genomic site which is cut and where the cell’s own DNA-repair machinery makes the final change utilizing a DNA template (single stranded or double stranded for larger edits) which is delivered together with the Cas9 enzyme. For this end, the first steps would be to define an objective precisely like make chlorophyll more sensitive to light or more prone to degradation under certain conditions, or reduce photoprotective quenching of the LHP and identify genes, regulatory regions or domains that are relevant for those functions. After that, decide what type of edit strategy is needed for a specific site (gene knockout, single or few nucleotide changes or sequence replacement) and design a guide RNA to bind to the target DNA of that specific site, and if a sequence rewrite is needed, design the DNA template for that repair which must have homologous endings that match those surrounding the cut site. To perform the actual edit the gRNA, Cas9 nuclease and template DNA are combined into a plasmid vector or ribonucleoprotein and introduced into the cells via heat shock, electroporation or lipofection. The limitations of this method might include off-target effects by binding and editing similar but not the exact sites; in a cell culture it might happen that not all cells be edited resulting in a mixture of edited and unedited cells, which can make it difficult to achieve a uniform result, and the HDR might have a low efficiency compared to the non-homologous end joining (NHEJ) pathway, not resulting in the intended edit.

First of all I used opentrons-art.rcdonovan.com to generate a base design with an image from “vecteezy.com” and then modified it manually to reach the final design

Then created the zones in red with the following logic (adapting from Example 5 and 7):

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for i in red:
    if i % 40 == 0:  ## Every 40 drops (not 20 because .5 drops), including at i == 0 for the start
     ## Aspirate the smaller value between pipette_20ul max_volume and how much volume is still needed given that each drop is a .5 drop
        pipette_20ul.aspirate(min(pipette_20ul.max_volume,(red.stop - i) * 0.5),
            location_of_color('Red'))
    dispense_and_detach(pipette_20ul, .5, cursor)
    cursor = cursor.move(types.Point(y=0, x=2.2))
    ## Here I start printing the red part of the cap in a side to side printer style movement — in an attempt to not have to enter every single coordinate and jumping the areas for other colors 
    if i == 13:
      cursor = cursor.move(types.Point(y=-2.2, x=2.2))
    if i > 13:
      cursor = cursor.move(types.Point(y=0, x=-4.4))
    if i  == 25:
      cursor = cursor.move(types.Point(y=0, x=-2.2*3))

Used Chat GPT to write all the coordinates using a “template dictionary” I wrote, and then adapted the logic I used before to work with the dictionary instead of a range:

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dots = list(yellow_stem.values())

  for i in range(len(dots)):
    if i % 40 == 0:
      ## same logic of refill but adapted to the yellow_stem dictionary
        pipette_20ul.aspirate(min(pipette_20ul.max_volume, (len(dots) - i) * 0.5),
            location_of_color('Orange'))
    cursor = center_location.move(dots[i])   
    dispense_and_detach(pipette_20ul, 0.5, cursor)

  pipette_20ul.drop_tip()

Repeated the same process for the green spots, but because I made some mistake in the red dots the coordinates from the green weren’t matching as they should, so I solved that by manually adjusting the red dots to the placement of the green spots